Regulated trafficking of cellulose synthases.

New findings reveal that proteins involved in cellulose biosynthesis undergo regulated trafficking between intracellular compartments and the plasma membrane. The coordinated secretion and internalization of these proteins involve both the actin and cortical microtubule cytoskeletons. This regulated trafficking allows the dynamic remodeling of cellulose synthase complex (CSC) secretion during cell expansion and differentiation. Several new actors of the cellulose synthesis machinery have been recently identified.

Temperature-sensitive alleles of RSW2 link the KORRIGAN endo-1,4-beta-glucanase to cellulose synthesis and cytokinesis in Arabidopsis.

An 8.5-kb cosmid containing the KORRIGAN gene complements the cellulose-deficient rsw2-1 mutant of Arabidopsis. Three temperature-sensitive alleles of rsw2 show single amino acid mutations in the putative endo-1,4-beta-glucanase encoded by KOR. The F1 from crosses between kor-1 and rsw2 alleles shows a weak, temperature-sensitive root phenotype. The shoots of rsw2-1 seedlings produce less cellulose and accumulate a short chain, readily extractable glucan resembling that reported for rsw1 (which is defective in a putative glycosyltransferase required for cellulose synthesis). The double mutant (rsw2-1 rsw1) shows further reductions in cellulose production relative to both single mutants, constitutively slow root growth, and enhanced temperature-sensitive responses that are typically more severe than in either single mutant. Abnormal cytokinesis and severely reduced birefringent retardation in elongating root cell walls of rsw2 link the enzyme to cellulose production for primary cell walls and probably cell plates. The Rsw2(-) phenotype generally resembles the Kor(-) and cellulose-deficient Rsw1(-) phenotypes, but anther dehiscence is impaired in Rsw2-1(-). The findings link a second putative enzyme activity to cellulose synthesis in primary cell walls of Arabidopsis and further increases the parallels to cellulose synthesis in Agrobacterium tumefaciens where the celA and celC genes are required and encode a putative glycosyltransferase and an endo-1,4-beta-glucanase related to RSW1 and KOR, respectively.

PROCUSTE1 encodes a cellulose synthase required for normal cell elongation specifically in roots and dark-grown hypocotyls of Arabidopsis.

Mutants at the PROCUSTE1 (PRC1) locus show decreased cell elongation, specifically in roots and dark-grown hypocotyls. Cell elongation defects are correlated with a cellulose deficiency and the presence of gapped walls. Map-based cloning of PRC1 reveals that it encodes a member (CesA6) of the cellulose synthase catalytic subunit family, of which at least nine other members exist in Arabidopsis. Mutations in another family member, RSW1 (CesA1), cause similar cell wall defects in all cell types, including those in hypocotyls and roots, suggesting that cellulose synthesis in these organs requires the coordinated expression of at least two distinct cellulose synthase isoforms.

Arabidopsis mutants impaired in cosuppression.

Post-transcriptional gene silencing (cosuppression) results in the degradation of RNA after transcription. A transgenic Arabidopsis line showing post-transcriptional silencing of a 35S-uidA transgene and uidA-specific methylation was mutagenized using ethyl methanesulfonate. Six independent plants were isolated in which uidA mRNA accumulation and beta-glucuronidase activity were increased up to 3500-fold, whereas the transcription rate of the 35S-uidA transgene was increased only up to threefold. These plants each carried a recessive monogenic mutation that is responsible for the release of silencing. These mutations defined two genetic loci, called sgs1 and sgs2 (for suppressor of gene silencing). Transgene methylation was distinctly modified in sgs1 and sgs2 mutants. However, methylation of centromeric repeats was not affected, indicating that sgs mutants differ from ddm (for decrease in DNA methylation) and som (for somniferous) mutants. Indeed, unlike ddm and som mutations, sgs mutations were not able to release transcriptional silencing of a 35S-hpt transgene. Conversely, both sgs1 and sgs2 mutations were able to release cosuppression of host Nia genes and 35S-Nia2 transgenes. These results therefore indicate that sgs mutations act in trans to impede specifically transgene-induced post-transcriptional gene silencing.

A plasma membrane-bound putative endo-1,4-beta-D-glucanase is required for normal wall assembly and cell elongation in Arabidopsis.

Endo-1,4-beta-D-glucanases (EGases) form a large family of hydrolytic enzymes in prokaryotes and eukaryotes. In higher plants, potential substrates in vivo are xyloglucan and non-crystalline cellulose in the cell wall. Gene expression patterns suggest a role for EGases in various developmental processes such as leaf abscission, fruit ripening and cell expansion. Using Arabidopsis thaliana genetics, we demonstrate the requirement of a specialized member of the EGase family for the correct assembly of the walls of elongating cells. KORRIGAN (KOR) is identified by an extreme dwarf mutant with pronounced architectural alterations in the primary cell wall. The KOR gene was isolated and encodes a membrane-anchored member of the EGase family, which is highly conserved between mono- and dicotyledonous plants. KOR is located primarily in the plasma membrane and presumably acts at the plasma membrane-cell wall interface. KOR mRNA was found in all organs examined, and in the developing dark-grown hypocotyl, mRNA levels were correlated with rapid cell elongation. Among plant growth factors involved in the control of hypocotyl elongation (auxin, gibberellins and ethylene) none significantly influenced KOR-mRNA levels. However, reduced KOR-mRNA levels were observed in det2, a mutant deficient for brassinosteroids. Although the in vivo substrate remains to be determined, the mutant phenotype is consistent with a central role for KOR in the assembly of the cellulose-hemicellulose network in the expanding cell wall.

The evolutionary analysis of the Tnt1 retrotransposon in Nicotiana species reveals the high variability of its regulatory sequences.

We studied the evolution of the tobacco Tnt1 retrotransposon by analyzing Tnt1 partial sequences containing both coding domains and U3 regulatory sequences obtained from a number of Nicotiana species. We detected three different subfamilies of Tnt1 elements, Tnt1A, Tnt1B, and Tnt1C, that differ completely in their U3 regions but share conserved flanking coding and LTR regions. U3 divergence between the three subfamilies is found in the region that contains the regulatory sequences that control the expression of the well-characterized Tnt1-94 element. This suggests that expression of the three Tnt1 subfamilies might be differently regulated. The three Tnt1 subfamilies were present in the Nicotiana genome at the time of species divergence, but have evolved independently since then in the different genomes. Each Tnt1 subfamily seems to have conserved its ability to transpose in a limited and different number of Nicotiana species. Our results illustrate the high variability of Tnt1 regulatory sequences. We propose that this high sequence variability could allow these elements to evolve regulatory mechanisms in order to optimize their coexistence with their host genome.

The promoter of the tobacco Tnt1 retrotransposon is induced by wounding and by abiotic stress.

The transcription of the tobacco Tnt1 retrotransposon was previously shown to be induced, in tobacco and in heterologous species, by microbial elicitors and by pathogen infections. We report here that the expression of the Tnt1 promoter is also activated in heterologous species such as tomato and Arabidopsis by wounding, freezing and by other abiotic factors known to induce the plant defence response, such as salicylic acid, CuCl2, or oxidative stress. A similar regulation is observed in tobacco for most treatments. The induction of the Tnt1 promoter expression by wounding remains localized around injury points. In CuCl2-treated Arabidopsis plants, the transcription of Tnt1 is correlated with accumulation of the phytoalexin camalexin and with the expression of the EL13 defence gene. The interest of the Tnt1 promoter as a sensitive indicator of the plant defence responses is discussed.

Quasispecies in retrotransposons: a role for sequence variability in Tnt1 evolution.

Retroviral replication is a very error-prone process. Replication of retroviruses gives rise to populations of closely related but different genomes referred to as 'quasispecies'. This huge swarm of different sequences constitutes a reservoir of potentially useful genomes in case of an environmental change, endowing retroviruses with extreme adaptability. Retrotransposons are mobile genetic elements closely related to retroviruses, and retrotransposition is as error prone as retroviral replication. The Tnt1 retrotransposon is present in hundreds of copies in the genome of tobacco that show a high level of sequence heterogeneity. When Tnt1 is expressed, its RNA is not a single sequence but a population of sequences displaying a quasispecies-like structure. This population structure gives to Tnt1, as in the case of retroviruses, a high sequence plasticity and an adaptive capacity. We propose this adaptivity as the major reason for Tnt1 maintenance in Nicotiana genomes and we discuss in this paper the importance of sequence variability for Tnt1 evolution.

The expression of the tobacco Tnt1 retrotransposon is linked to plant defense responses.

Activation of retrotransposons by stresses and external changes is common in all eukaryotic systems, including plants. The transcription of the tobacco Tnt1 retrotransposon was studied in its natural host as well as in Arabidopsis and tomato. It is activated by factors of microbial origin, by external stresses, and by viral, bacterial, and fungal attacks. Tnt1 expression is linked with the biological responses of the plant to the elicitor or to the pathogen attack and in particular with the early steps of the metabolic pathways leading to the activation of plant defense genes. In most cases, the basic features of Tnt1 regulation in tobacco are maintained in tomato and Arabidopsis, but some host-specific regulations were shown. The U3 region of the Tnt1 LTR contains the major cis-acting components of Tnt1 transcriptional activation in association with the plant defense responses. Furthermore, the Tnt1 U3 region, and especially the tandemly repeated BII boxes, contains several sequences similar to well-characterized motifs involved in the activation of several plant defense genes. The possible origin of Tnt1 regulatory sequences as well as the biological implications of Tnt1 activation by pathogen attacks are discussed.

In vivo characterization of transcriptional regulatory sequences involved in the defence-associated expression of the tobacco retrotransposon Tnt1.

The expression of the tobacco retrotransposon Tnt1 is induced by wounding, pathogen infections as well as microbial elicitors and abiotic factors known to induce the plant defence response. We report here that the LTR U3 region is sufficient to mediate transcriptional activation by biotic and abiotic elicitors in stable transgenic conditions. We have used in vivo footprinting techniques in order to analyse the cis-regulatory elements of the LTR U3 region that mediate the induction of Tnt1 expression. Our results indicate that a tandemly repeated short element, named BII box, is involved in the transcriptional activation of the tobacco retrotransposon Tnt1 in association with the plant defence signaling cascade.

Sequence variability within the tobacco retrotransposon Tnt1 population.

Retroviruses consist of populations of different but closely related genomes referred to as quasispecies. A high mutation rate coupled with extremely rapid replication cycles allows these sequences to be highly interconnected in a rapid equilibrium. It is not known if other retroelements can show a similar population structure. We show here that when the tobacco Tnt1 retrotransposon is expressed, its RNA is not a unique sequence but a population of different but closely related sequences. Nevertheless, this highly variable population is not in a rapid equilibrium and could not be considered as a quasispecies. We have thus named the structure presented by Tnt1 RNA quasispecies-like. We show that the expression of Tnt1 in different situations gives rise to different populations of Tnt1 RNA sequences, suggesting an adaptive capacity for this element. The analysis of the variability within the total genomic population of Tnt1 elements shows that mutations frequently occur in important regulatory elements and that defective elements are often produced. We discuss the implications that this population structure could have for Tnt1 regulation and evolution.