KORRIGAN1 Research Articles

Overexpression of the bamboo sucrose synthase gene (BeSUS5) improves cellulose production, cell wall thickness and fiber quality in transgenic poplar

In plants, sucrose synthase (SUS, EC 2.4.1.13) is widely considered a multifunctional protein involved in modulating sink strength, cellulose biosynthesis, and carbon partitioning. However, supporting genetic evidence regarding the role of SUS from bamboo in fiber development is lacking. Here, we obtained transgenic poplar lines overexpressing the bamboo BeSUS5 gene and conducted functional analysis. We found that overexpression of BeSUS5 enhanced the activity of SUS and significantly promoted the growth of the plants, especially xylem growth. In BeSUS5 overexpressed poplar plants, the total soluble sugar (TSS) and starch contents were decreased in leaves, while the cellulose content was increased in stems, indicating that overexpression of BeSUS5 might enhance the partitioning of carbon to cellulose in poplar. Consistent with these results, the expression of cellulose biosynthesis and phloem loading–related genes, such as cellulose synthase (CesA7), KORRIGAN (KOR), and sucrose transporter (SUT1), was upregulated in transgenic plants. As a result, transgenic poplars displayed not only an increase in cell wall thickness and cell wall crystallinity but also an altered stem fiber phenotype. Taken together, our results imply the vast potential of BeSUS5 for the genetic improvement of wood cellulose production and fiber quality.

Lack of endoplasmic reticulum quality control (ERQC) promotes tonoplast (TP) targeting of KORRIGAN 1 (KOR1)

ABSTRACTCellular dynamics of KORRIGAN 1 (KOR1) is closely linked with cellulose biosynthesis and plant osmotic stress tolerance. Cycling of KOR1 between the plasma membrane (PM) and trans-Golgi Network (TGN) is maintained by sequence motifs and protein structures that are recognized by cellular transport and quality control mechanisms. Several mutations in KOR1, as well as in the host genetic background, promote the mistargeting of KOR1 and induce KOR1 accumulation in the tonoplast (TP). Yet, little is known about how retention and sorting of KOR1 are regulated in the PM-TGN cycle. Forward genetic screening for GFP-KOR1 mislocalizing phenotype resulted in several mutant lines with different localization patterns or signal intensity of GFP-KOR1. One of the identified mutants were disrupted at UDP-glucose:glycoprotein glucosyltransferase (UGGT) locus, which is essential for the protein quality control in the ER. Our finding suggests the mis/unfolded structure of KOR1 triggers the TP targeting.

Multiple Quality Control Mechanisms in the ER and TGN Determine Subcellular Dynamics and Salt-Stress Tolerance Function of KORRIGAN1.

Among many glycoproteins within the plant secretory system, KORRIGAN1 (KOR1), a membrane-anchored endo-β-1,4-glucanase involved in cellulose biosynthesis, provides a link between N-glycosylation, cell wall biosynthesis, and abiotic stress tolerance. After insertion into the endoplasmic reticulum, KOR1 cycles between the trans-Golgi network (TGN) and the plasma membrane (PM). From the TGN, the protein is targeted to growing cell plates during cell division. These processes are governed by multiple sequence motifs and also host genotypes. Here, we investigated the interaction and hierarchy of known and newly identified sorting signals in KOR1 and how they affect KOR1 transport at various stages in the secretory pathway. Conventional steady-state localization showed that structurally compromised KOR1 variants were directed to tonoplasts. In addition, a tandem fluorescent timer technology allowed for differential visualization of young versus aged KOR1 proteins, enabling the analysis of single-pass transport through the secretory pathway. Observations suggest the presence of multiple checkpoints/branches during KOR1 trafficking, where the destination is determined based on KOR1's sequence motifs and folding status. Moreover, growth analyses of dominant PM-confined KOR1-L48L49→A48A49 variants revealed the importance of active removal of KOR1 from the PM during salt stress, which otherwise interfered with stress acclimation.

Arabidopsis thaliana KORRIGAN1 protein: N-glycan modification, localization, and function in cellulose biosynthesis and osmotic stress responses

Plant cellulose biosynthesis is a complex process involving cellulose-synthase complexes (CSCs) and various auxiliary factors essential for proper orientation and crystallinity of cellulose microfibrils in the apoplast. Among them is KORRIGAN1 (KOR1), a type-II membrane protein with multiple N-glycans within its C-terminal cellulase domain. N-glycosylation of the cellulase domain was important for KOR1 targeting to and retention within the trans-Golgi network (TGN), and prevented accumulation of KOR1 at tonoplasts. The degree of successful TGN localization of KOR1 agreed well with in vivo-complementation efficacy of the rsw2–1 mutant, suggesting non-catalytic functions in the TGN. A dynamic interaction network involving microtubules, CSCs, KOR1, and currently unidentified glycoprotein component(s) likely determines stress-triggered re-organization of cellulose biosynthesis and resumption of cell-wall growth under stress.

Down-regulation of the cotton endo-1,4-β-glucanase gene KOR1 disrupts endosperm cellularization, delays embryo development, and reduces early seedling vigour.

Towards the aim of examining the potential function of KORRIGAN (KOR), a highly conserved membrane-bound endoglucanase, in reproductive development, here transgenic evidence is provided that a cotton (Gossypium hirsutum) endoglucanase, GhKOR1, plays significant roles in endosperm and embryo development. RNA interference (RNAi)- and co-suppression-mediated down-regulation of GhKOR1 resulted in smaller filial tissue and reduced seed weight, which were characterized by disrupted endosperm cellularization and delayed embryo development, leading to a delayed germination and a weak growth of seedlings early in development. The transgenic seeds exhibited fewer and smaller endosperm cells with irregular and brittle cell walls, and their embryos developed only to the globular stage at 10 days post-anthesis (DPA) when the wild-type endosperm has become highly cellularized and the embryo has progressed to the heart stage. The transgenic seed also displayed a significant reduction of callose in the seed coat transfer cells and reduced cellulose content both in the seed coat and in mature fibres. These findings demonstrate that GhKOR1 is required for the developmental of both seed filial and maternal tissues and the establishment of seedling vigour.

CESA TRAFFICKING INHIBITOR inhibits cellulose deposition and interferes with the trafficking of cellulose synthase complexes and their associated proteins KORRIGAN1 and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1.

Cellulose synthase complexes (CSCs) at the plasma membrane (PM) are aligned with cortical microtubules (MTs) and direct the biosynthesis of cellulose. The mechanism of the interaction between CSCs and MTs, and the cellular determinants that control the delivery of CSCs at the PM, are not yet well understood. We identified a unique small molecule, CESA TRAFFICKING INHIBITOR (CESTRIN), which reduces cellulose content and alters the anisotropic growth of Arabidopsis (Arabidopsis thaliana) hypocotyls. We monitored the distribution and mobility of fluorescently labeled cellulose synthases (CESAs) in live Arabidopsis cells under chemical exposure to characterize their subcellular effects. CESTRIN reduces the velocity of PM CSCs and causes their accumulation in the cell cortex. The CSC-associated proteins KORRIGAN1 (KOR1) and POM2/CELLULOSE SYNTHASE INTERACTIVE PROTEIN1 (CSI1) were differentially affected by CESTRIN treatment, indicating different forms of association with the PM CSCs. KOR1 accumulated in bodies similar to CESA; however, POM2/CSI1 dissociated into the cytoplasm. In addition, MT stability was altered without direct inhibition of MT polymerization, suggesting a feedback mechanism caused by cellulose interference. The selectivity of CESTRIN was assessed using a variety of subcellular markers for which no morphological effect was observed. The association of CESAs with vesicles decorated by the trans-Golgi network-localized protein SYNTAXIN OF PLANTS61 (SYP61) was increased under CESTRIN treatment, implicating SYP61 compartments in CESA trafficking. The properties of CESTRIN compared with known CESA inhibitors afford unique avenues to study and understand the mechanism under which PM-associated CSCs are maintained and interact with MTs and to dissect their trafficking routes in etiolated hypocotyls.

Multiple N-glycans cooperate in the subcellular targeting and functioning of Arabidopsis KORRIGAN1.

Arabidopsis thaliana KORRIGAN1 (KOR1) is an integral membrane endo-β1,4-glucanase in the trans-Golgi network and plasma membrane that is essential for cellulose biosynthesis. The extracellular domain of KOR1 contains eight N-glycosylation sites, N1 to N8, of which only N3 to N7 are highly conserved. Genetic evidence indicated that cellular defects in attachment and maturation of these N-glycans affect KOR1 function in vivo, whereas the manner by which N-glycans modulate KOR1 function remained obscure. Site-directed mutagenesis analysis of green fluorescent protein (GFP)-KOR1 expressed from its native regulatory sequences established that all eight N-glycosylation sites (N1 to N8) are used in the wild type, whereas stt3a-2 cells could only inefficiently add N-glycans to less conserved sites. GFP-KOR1 variants with a single N-glycan at nonconserved sites were less effective than those with one at a highly conserved site in rescuing the root growth phenotype of rsw2-1 (kor1 allele). When functionally compromised, GFP-KOR1 tended to accumulate at the tonoplast. GFP-KOR1Δall (without any N-glycan) exhibited partial complementation of rsw2-1; however, root growth of this line was still negatively affected by the absence of complex-type N-glycan modifications in the host plants. These results suggest that one or several additional factor(s) carrying complex N-glycans cooperate(s) with KOR1 in trans to grant proper targeting/functioning in plant cells.

Cellulase in Cellulose Synthase: A Cat among the Pigeons?

Arabidopsis ( Arabidopsis thaliana ) KORRIGAN1 (KOR1) and its close relatives are endo-1,4-β-d-glucanases whose hydrolyzing activity on amorphous cellulose has been demonstrated ([Molhoj et al., 2001][1]; [Master et al., 2004][2]; [Liebminger et al., 2013][3]). An outstanding characteristic of

Absence of endo-1,4-β-glucanase KOR1 alters the Jasmonate-dependent defence response to Pseudomonas syringae in Arabidopsis

During plant–pathogen interactions, the plant cell wall forms part of active defence against invaders. In recent years, cell wall-editing enzymes, associated with growth and development, have been related to plant susceptibility or resistance. Our previous work identified a role for several tomato and Arabidopsis endo-1,4-β-glucanases (EGs) in plant–pathogen interactions. Here we studied the response of the Arabidopsis thaliana T-DNA insertion mutant lacking EG Korrigan1 (KOR1) infected with Pseudomonas syringae. KOR1 is predicted to be an EG which is thought to participate in cellulose biosynthesis. We found that kor1-1 plants were more susceptible to P. syringae, and displayed severe disease symptoms and enhanced bacterial growth if compared to Wassilewskija (Ws) wild-type plants. Hormonal and gene expression analyses revealed that the jasmonic acid (JA) pathway was activated more in kor1-1 plants with an increase in the JA-biosynthesis gene LOX3 and a greater accumulation of JA. Upon infection the accumulation of JA and JA-isoleucine (JA-Ile) was higher than in wild-type plants and increased the induction of LOX3 and the JA-responsive PDF1.2 gene. In addition, the increase of salicylic acid (SA) in healthy and infected kor1-1 may reflect the complex interaction between JA and SA, which results in the more susceptible phenotype displayed by the infected mutant plants. Callose deposition was enhanced in infected kor1-1 and an increase in pathogen-induced hydrogen peroxide took place. The susceptible phenotype displayed by KOR1-deficient plants was coronatine-independent. No significant changes were detected in the hormonal profile of the kor1-1 plants infected by coronatine-deficient P. syringae cmaA, which supports that absence of EG KOR1 alters per se the plant response to infection. We previously reported increased resistance of kor1-1 to B. cinerea, hence, the lack of this EG alters cell wall properties and plant responses in such a way that benefits P. syringae colonisation but restricts B. cinerea invasion.

The Cellulase KORRIGAN Is Part of the Cellulose Synthase Complex.

Plant growth and organ formation depend on the oriented deposition of load-bearing cellulose microfibrils in the cell wall. Cellulose is synthesized by a large relative molecular weight cellulose synthase complex (CSC), which comprises at least three distinct cellulose synthases. Cellulose synthesis in plants or bacteria also requires the activity of an endo-1,4-β-d-glucanase, the exact function of which in the synthesis process is not known. Here, we show, to our knowledge for the first time, that a leaky mutation in the Arabidopsis (Arabidopsis thaliana) membrane-bound endo-1,4-β-d-glucanase KORRIGAN1 (KOR1) not only caused reduced CSC movement in the plasma membrane but also a reduced cellulose synthesis inhibitor-induced accumulation of CSCs in intracellular compartments. This suggests a role for KOR1 both in the synthesis of cellulose microfibrils and in the intracellular trafficking of CSCs. Next, we used a multidisciplinary approach, including live cell imaging, gel filtration chromatography analysis, split ubiquitin assays in yeast (Saccharomyces cerevisiae NMY51), and bimolecular fluorescence complementation, to show that, in contrast to previous observations, KOR1 is an integral part of the primary cell wall CSC in the plasma membrane.

PtrKOR1 is required for secondary cell wall cellulose biosynthesis in Populus

KORRIGAN (KOR), encoding an endo-1,4-β-glucanase, plays a critical role in the cellulose synthesis of plant cell wall formation. KOR sequence orthologs are duplicated in the Populus genome relative to Arabidopsis. This study reports an expression analysis of the KOR genes in Populus. The five PtrKOR genes displayed different expression patterns, suggesting that they play roles in different developmental processes. Through RNAi suppression, results demonstrated that PtrKOR1 is required for secondary cell wall cellulose formation in Populus. Together, the results suggest that the PtrKOR genes may play distinct roles in association with cell wall formation in different tissues.

Characterizing the Link between Glycosylation State and Enzymatic Activity of the Endo-β1,4-glucanase KORRIGAN1 from Arabidopsis thaliana

Defects in N-glycosylation and N-glycan processing frequently cause alterations in plant cell wall architecture, including changes in the structure of cellulose, which is the most abundant plant polysaccharide. KORRIGAN1 (KOR1) is a glycoprotein enzyme with an essential function during cellulose biosynthesis in Arabidopsis thaliana. KOR1 is a membrane-anchored endo-β1,4-glucanase and contains eight potential N-glycosylation sites in its extracellular domain. Here, we expressed A. thaliana KOR1 as a soluble, enzymatically active protein in insect cells and analyzed its N-glycosylation state. Structural analysis revealed that all eight potential N-glycosylation sites are utilized. Individual elimination of evolutionarily conserved N-glycosylation sites did not abolish proper KOR1 folding, but mutations of Asn-216, Asn-324, Asn-345, and Asn-567 resulted in considerably lower enzymatic activity. In contrast, production of wild-type KOR1 in the presence of the class I α-mannosidase inhibitor kifunensine, which abolished the conversion of KOR1 N-glycans into complex structures, did not affect the activity of the enzyme. To address N-glycosylation site occupancy and N-glycan composition of KOR1 under more natural conditions, we expressed a chimeric KOR1-Fc-GFP fusion protein in leaves of Nicotiana benthamiana. Although Asn-108 and Asn-133 carried oligomannosidic N-linked oligosaccharides, the six other glycosylation sites were modified with complex N-glycans. Interestingly, the partially functional KOR1 G429R mutant encoded by the A. thaliana rsw2-1 allele displayed only oligomannosidic structures when expressed in N. benthamiana, indicating its retention in the endoplasmic reticulum. In summary, our data indicate that utilization of several N-glycosylation sites is important for KOR1 activity, whereas the structure of the attached N-glycans is not critical.

The endo‐1,4‐β‐glucanase Korrigan exhibits functional conservation between gymnosperms and angiosperms and is required for proper cell wall formation in gymnosperms

The evolution of compositional polymers and their complex arrangement and deposition in the cell walls of terrestrial plants included the acquisition of key protein functions. A membrane-bound endoglucanase, termed Korrigan (KOR), has been shown to be required for proper cellulose synthesis. To date, no extensive characterization of the gymnosperm KOR has been undertaken. Characterization of the white spruce (Picea glauca) gene encoding KOR (PgKOR) shows conserved protein features such as polarized targeting signals and residues predicted to be essential for catalytic activity. The rescue of the Arabidopsis thaliana kor1-1 mutant by the expression of PgKOR suggests gene conservation, providing evidence for functional equivalence. Analyses of endogenous KOR expression in white spruce revealed the highest expression in young developing tissues, which corresponds with primary cell wall development. Additionally, RNA interference of the endogenous gymnosperm gene substantially reduced growth and structural glucose content, but had no effect on cellulose ultrastructure. Partial functional conservation of KOR in gymnosperms suggests that its role in cell wall synthesis dates back to 300 million yr ago (Mya), predating angiosperms, which arose 130 Mya, and shows that proteins contributing to proper cellulose deposition are important conserved features of vascular plants.

The trafficking and behavior of cellulose synthase and a glimpse of potential cellulose synthesis regulators

Cellulose biosynthesis is a topic of intensive research not only due to the significance of cellulose in the integrity of plant cell walls, but also due to the potential of using cellulose, a natural carbon source, in the production of biofuels. Characterization of the composition, regulation, and trafficking of cellulose synthase complexes (CSCs) is critical to an understanding of cellulose biosynthesis as well as the characterization of additional proteins that contribute to the production of cellulose either through direct interactions with CSCs or through indirect mechanisms. In this review, a highlight of a few proteins that appear to affect cellulose biosynthesis, which includes: KORRIGAN (KOR), Cellulose Synthase-Interactive Protein 1 (CSI1), and the poplar microtubule-associated protein, PttMAP20, will accompany a description of cellulose synthase (CESA) behavior and a discussion of CESA trafficking compartments that might act in the regulation of cellulose biosynthesis.

KORRIGAN1 and its Aspen Homolog PttCel9A1 Decrease Cellulose Crystallinity in Arabidopsis Stems

KORRIGAN1 (KOR1) is a membrane-bound cellulase implicated in cellulose biosynthesis. PttCel9A1 from hybrid aspen (Populus tremula L. x tremuloides Michx.) has high sequence similarity to KOR1 and we demonstrate here that it complements kor1-1 mutants, indicating that it is a KOR1 ortholog. We investigated the function of PttCel9A1/KOR1 in Arabidopsis secondary growth using transgenic lines expressing 35S::PttCel9A1 and the KOR1 mutant line irx2-2. The presence of elevated levels of PttCel9A1/KOR1 in secondary walls of 35S::PttCel9A1 lines was confirmed by in muro visualization of cellulase activity. Compared with the wild type, 35S::PttCel9A1 lines had higher trifluoroacetic acid (TFA)-hydrolyzable glucan contents, similar Updegraff cellulose contents and lower cellulose crystallinity indices, as determined by (13)C solid-state nuclear magnetic resonance (NMR) spectroscopy. irx2-2 mutants had wild-type TFA-hydrolyzable glucan contents, but reduced Updegraff cellulose contents and higher than wild-type cellulose crystallinity indices. The data support the hypothesis that PttCel9A1/KOR1 activity is present in cell walls, where it facilitates cellulose biosynthesis in a way that increases the amount of non-crystalline cellulose.

Carbohydrate-Binding Module of a Rice Endo-β-1,4-glycanase, OsCel9A , Expressed in Auxin-Induced Lateral Root Primordia, is Post-Translationally Truncated

We report the cloning of a glycoside hydrolase family (GHF) 9 gene of rice (Oryza sativa L. cv. Sasanishiki), OsCel9A, corresponding to the auxin-induced 51 kDa endo-1,4-beta-glucanase (EGase). This enzyme reveals a broad substrate specificity with respect to sugar backbones (glucose and xylose) in beta-1,4-glycans of type II cell wall. OsCel9A encodes a 640 amino acid polypeptide and is an ortholog of TomCel8, a tomato EGase containing a carbohydrate-binding module (CBM) 2 sequence at its C-terminus. The expression of four rice EGase genes including OsCel9A showed different patterns of organ specificity and responses to auxin. OsCel9A was preferentially expressed during the initiation of lateral roots or subcultured root calli, but was hardly expressed during auxin-induced coleoptile elongation or in seed calli, in contrast to OsCel9D, a KORRIGAN (KOR) homolog. In situ localization of OsCel9A transcripts demonstrated that its expression was specifically up-regulated in lateral root primordia (LRP). Northern blotting analysis showed the presence of a single product of OsCel9A. In contrast, both mass spectrometric analyses of peptide fragments from purified 51 kDa EGase proteins and immunogel blot analysis of EGase proteins in root extracts using two antibodies against internal peptide sequences of OsCel9A revealed that the entire CBM2 region was post-translationally truncated from the 67 kDa nascent protein to generate 51 kDa EGase isoforms. Analyses of auxin concentration and time course dependence of accumulation of two EGase isoforms suggested that the translation and post-translational CBM2 truncation of the OsCel9A gene may participate in lateral root development.

AnArabidopsisEndo-1,4-β-d-Glucanase Involved in Cellulose Synthesis Undergoes Regulated Intracellular Cycling[W

The synthesis of cellulose microfibrils requires the presence of a membrane-bound endo-1,4-beta-D-glucanase, KORRIGAN1 (KOR1). Although the exact biochemical role of KOR1 in cellulose synthesis is unknown, we used the protein as a marker to explore the potential involvement of subcellular transport processes in cellulose synthesis. Using immunofluorescence and a green fluorescent protein (GFP)-KOR1 fusion that complemented the phenotype conferred by the kor1-1 mutant, we investigated the distribution of KOR1 in epidermal cells in the root meristem. KOR1 was localized in intracellular compartments corresponding to a heterogeneous population of organelles, which comprised the Golgi apparatus, FM4-64-labeled compartments referred to as early endosomes, and, in the case of GFP-KOR1, the tonoplast. Inhibition of cellulose synthesis by isoxaben promoted a net redistribution of GFP-KOR1 toward a homogeneous population of compartments, distinct from early endosomes, which were concentrated close to the plasma membrane facing the root surface. A redistribution of GFP-KOR1 away from early endosomes was also observed in the same cells at later stages of cell elongation. A subpopulation of GFP-KOR1-containing compartments followed trajectories along the plasma membrane, and this motility required intact microtubules. These observations demonstrate that the deposition of cellulose, like chitin synthesis in yeast, involves the regulated intracellular cycling of at least one enzyme required for its synthesis.

Virus-induced gene silencing as a tool for functional genomics in a legume species.

Virus-induced gene silencing (VIGS) is an attractive reverse-genetics tool for studies of gene function. However, efficient VIGS has only been accomplished in a few plant species. In order to extend the application of VIGS, we examined whether a VIGS vector based on Pea early browning virus (PEBV) would produce recognizable phenotypes in Pisum sativum. A plasmid vector of PEBV was modified to allow agro-inoculation and insertion of heterologous sequences. cDNA fragments of the P. sativum phytoene desaturase (PDS), LEAFY (LFY) and KORRIGAN1 (KOR1) homologues were inserted into the PEBV RNA2 vector, replacing the genes required for nematode transmission. Pisum sativum inoculated with PEBV carrying a fragment of PsPDS developed characteristic photo-bleached leaves and this phenotype was associated with a significant reduction in PsPDS mRNA. The P. sativum homologue of LFY is known as UNIFOLIATA (UNI). Plants inoculated with PEBV carrying a fragment of UNI developed distorted flowers and leaves with modified architecture, which are also observed in UNI-mutants. In Arabidopsis thaliana, the KOR1-mutant is characterized by an extreme dwarf phenotype. Pisum sativum plants inoculated with PEBV carrying a fragment of PsKOR1 displayed a significant reduction in height and inhibition of root growth. The PEBV VIGS vector did not affect the ability of P. sativum to flower, set seeds, and form nodules characteristic of symbiosis with rhizobium. These results suggest that the PEBV vector can be applied to functional genomics in a legume species to study genes involved in a wide range of biological processes.

The irregular xylem 2 mutant is an allele of korrigan that affects the secondary cell wall of Arabidopsis thaliana.

The irregular xylem 2 (irx2) mutant of Arabidopsis thaliana exhibits a cellulose deficiency in the secondary cell wall, which is brought about by a point mutation in the KORRIGAN (KOR) beta,1-4 endoglucanase (beta,1-4 EGase) gene. Measurement of the total crystalline cellulose in the inflorescence stem indicates that the irx2 mutant contains approximately 30% of the level present in the wild type (WT). Fourier-Transform Infra Red (FTIR) analysis, however, indicates that there is no decrease in cellulose in primary cell walls of the cortical and epidermal cells of the stem. KOR expression is correlated with cellulose synthesis and is highly expressed in cells synthesising a secondary cell wall. Co-precipitation experiments, using either an epitope-tagged form of KOR or IRX3 (AtCesA7), suggest that KOR is not an integral part of the cellulose synthase complex. These data are supported by immunolocalisation of KOR that suggests that KOR does not localise to sites of secondary cell wall deposition in the developing xylem. The defect in irx2 plant is consistent with a role for KOR in the later stages of secondary cell wall formation, suggesting a role in processing of the growing microfibrils or release of the cellulose synthase complex.

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.