SummaryFruit development is usually triggered by ovule fertilization, and it requires coordination between seed development and the growth and differentiation of the ovary to host the seeds. Hormones are known to synchronize these two processes, but the role of each hormone, and the mechanism by which they interact, are still unknown. Here we show that auxin and gibberellins (GAs) act in a hierarchical scheme. The synthetic reporter construct DR5:GFP showed that fertilization triggered an increase in auxin response in the ovules, which could be mimicked by blocking polar auxin transport. As the application of GAs did not affect auxin response, the most likely sequence of events after fertilization involves auxin-mediated activation of GA synthesis. We have confirmed this, and have shown that GA biosynthesis upon fertilization is localized specifically in the fertilized ovules. Furthermore, auxin treatment caused changes in the expression of GA biosynthetic genes similar to those triggered by fertilization, and also restricted to the ovules. Finally, GA signaling was activated in ovules and valves, as shown by the rapid downregulation of the fusion protein RGA-GFP after pollination and auxin treatment. Taken together, this evidence suggests a model in which fertilization would trigger an auxin-mediated promotion of GA synthesis specifically in the ovule. The GAs synthesized in the ovules would be then transported to the valves to promote GA signaling and thus coordinate growth of the silique.

SummaryDELLA proteins are plant nuclear factors that restrain growth and proliferation in response to hormonal signals. The effects of the manipulation of the DELLA pathway in the making of a berry-like fruit were investigated. The expression of the Arabidopsis thaliana gain-of-function DELLA allele Atgai del in tomato (Solanum lycopersicum L.) produced partially sterile dwarf plants and compacted influorescences, as expected for a constitutively activated growth repressor. In contrast, antisense silencing of the single endogenous tomato DELLA gene homologue (SlDELLA) produced slender-like plants with elongated flower trusses. Interestingly, the depletion of SlDELLA in tomato was sufficient to overcome the growth arrest normally imposed on the ovary at anthesis, resulting in parthenocarpic fruits in the absence of pollination. Antisense SlDELLA-engineered fruits were smaller in size and elongated in shape compared with wild type. Cell number estimations showed that fruit set, resulting from reduced SlDELLA expression, arose from activated cell elongation at the longitudinal and lateral axes of the fruit pericarp, bypassing phase-II (post-pollination) cell divisions. Parthenocarpy caused by SlDELLA depletion is facultative, as hand pollination restored wild-type fruit phenotype. This indicates that fertilization-associated SlDELLA-independent signals are operational in ovary-fruit transitions. SlDELLA was also found to restrain growth in other reproductive structures, affecting style elongation, stylar hair primordial growth and stigma development.

Cell size and secondary cell wall patterning are crucial for the proper functioning of xylem vessel elements in the vascular tissues of plants. Through detailed anatomical characterization of Arabidopsis thaliana hypocotyls, we observed that mutations in the putative spermine biosynthetic gene ACL5 severely affected xylem specification: the xylem vessel elements of the acl5 mutant were small and mainly of the spiral type, and the normally predominant pitted vessels as well as the xylem fibers were completely missing. The cell-specific expression of ACL5 in the early developing vessel elements, as detected by in situ hybridization and reporter gene analyses, suggested that the observed xylem vessel defects were caused directly by the acl5 mutation. Exogenous spermine prolonged xylem element differentiation and stimulated cell expansion and cell wall elaboration in xylogenic cell cultures of Zinnia elegans, suggesting that ACL5 prevents premature death of the developing vessel elements to allow complete expansion and secondary cell wall patterning. This was further supported by our observations that the vessel elements of acl5 seemed to initiate the cell death program too early and that the xylem defects associated with acl5 could be largely phenocopied by induction of premature, diphtheria toxin-mediated cell death in the ACL5-expressing vessel elements. We therefore provide, for the first time, mechanistic evidence for the function of ACL5 in xylem specification through its action on the duration of xylem element differentiation.

The conversion of putrescine to spermidine in the biosynthetic pathway of plant polyamines is catalyzed by two closely related spermidine synthases, SPDS1 and SPDS2, in Arabidopsis. In the yeast two-hybrid system, SPDS2 was found to interact with SPDS1 and a novel protein, SPMS (spermine synthase), which is homologous with SPDS2 and SPDS1. SPMS interacts with both SPDS1 and SPDS2 in yeast and in vitro. Unlike SPDS1 and SPDS2, SPMS failed to suppress the spe ⌬ 3 deficiency of spermidine synthase in yeast. However, SPMS was able to complement the spe ⌬ 4 spermine deficiency in yeast, indicating that SPMS is a novel spermine synthase. The SPDS and SPMS proteins showed no homodimerization but formed heterodimers in vitro. Pairwise coexpression of hemagglutinin-and c-Myc epitope-labeled proteins in Arabidopsis cells confirmed the existence of coimmunoprecipitating SPDS1-SPDS2 and SDPS2-SPMS heterodimers in vivo. The epitope-labeled SPDS and SPMS proteins copurified with protein complexes ranging in size from 650 to 750 kD. Our data demonstrate the existence of a metabolon involving at least the last two steps of polyamine biosynthesis in Arabidopsis.

Mitogen-activated protein kinase (MAPK) cascades play an important role in mediating stress responses in plants. In Arabidopsis, 20 MAPKs have been identified and classified into four major groups (A-D). Little is known about the role of group C MAPKs. We have studied the activation of Arabidopsis subgroup C1 MAPKs (AtMPK1/AtMPK2) in response to mechanical injury. An increase in their kinase activity was detected in response to wounding that was blocked by cycloheximide. Jasmonic acid (JA) activated AtMPK1/AtMPK2 in the absence of wounding. Wound and JA-induction of AtMPK1/2 kinase activity was not prevented in the JA-insensitive coi1 mutant. Other stress signals, such as abscisic acid (ABA) and hydrogen peroxide, activated AtMPK1/2. This report shows for the first time that regulation of AtMPK1/2 kinase activity in Arabidopsis might be under the control of signals involved in different kinds of stress.

Control of tissue dimensions in multicellular organisms requires the precise quantitative regulation of mitotic activity. In plants, where cells are immobile, tissue size is achieved through control of both cell division orientation and mitotic rate. The bHLH transcription factor heterodimer formed by target of monopteros5 (TMO5) and lonesome highway (LHW) is a central regulator of vascular width-increasing divisions. An important unanswered question is how its activity is limited to specify vascular tissue dimensions. Here we identify a regulatory network that restricts TMO5/LHW activity. We show that thermospermine synthase ACAULIS5 antagonizes TMO5/LHW activity by promoting the accumulation of SAC51-LIKE (SACL) bHLH transcription factors. SACL proteins heterodimerize with LHW-therefore likely competing with TMO5/LHW interactions-prevent activation of TMO5/LHW target genes, and suppress the over-proliferation caused by excess TMO5/LHW activity. These findings connect two thus-far disparate pathways and provide a mechanistic understanding of the quantitative control of vascular tissue growth.

Polyamine biosynthesis is an ancient metabolic pathway present in all organisms. Aminopropyltransferases are key enzymes that mediate the synthesis of spermidine, spermine, and thermospermine. The relatively high sequence similarity between aminopropyltransferases and their similarity with putrescine N-methyltransferases (PMT) raises the question of whether they share a common ancestor or have evolved by convergence. Here we show that aminopropyltransferases and PMT are phylogenetically interconnected, and the different activities have been generated by unusually frequent events of diversification of existing functions. Although all spermidine synthases (SPDSs) derive from a common ancestor preceding the separation between prokaryotes and eukaryotes, they have been the origin of a variety of new activities. Among those, spermine synthases (SPMSs) represent a novelty independently arisen at least 3 times, in animals, fungi, and plants. The most parsimonious mechanism would involve the duplication and change of function of preexisting SPDS genes in each phylum. Although spermine is not essential for life, the repeated invention of SPMS and its conservation strongly argues for an evolutionary advantage derived from its presence. Moreover, the appearance of thermospermine synthase (tSPMS) in several genera of Archaea and Bacteria was accompanied by a loss of SPDS, suggesting that the new activity originated as a change of function of this enzyme. Surprisingly, tSPMS was later acquired by plants at an early stage of evolution by horizontal gene transfer and has proven to be essential for vascular development in tracheophytes. Finally, the synthesis of nicotine and tropane alkaloids in Solanales was favored by the origination of a new activity, PMT, as a duplication and change of function from SPDS.

Polyamines are small ubiquitous molecules that have been involved in nearly all developmental processes, including the stress response. Nevertheless, no direct evidence of a role of polyamines in the wound response has been described. We have studied the expression of genes involved in polyamine biosynthesis in response to mechanical injury. An increase in the expression of the arginine decarboxylase 2 (ADC2) gene in response to mechanical wounding and methyl jasmonate (JA) treatment in Arabidopsis was detected by using DNA microarray and RNA gel-blot analysis. No induction was observed for the ADC1 gene or other genes coding for spermidine and spermine synthases, suggesting that ADC2 is the only gene of polyamine biosynthesis involved in the wounding response mediated by JA. A transient increase in the level of free putrescine followed the increase in the mRNA level for ADC2. A decrease in the level of free spermine, coincident with the increase in putrescine after wounding, was also observed. Abscisic acid effected a strong induction on ADC2 expression and had no effect on ADC1 expression. Wound-induction of ADC2 mRNA was not prevented in the JA-insensitive coi1 mutant. The different pattern of expression of ADC2 gene in wild-type and coi1 mutant might be due to the dual regulation of ADC2 by abscisic acid and JA signaling pathways. This is the first direct evidence of a function of polyamines in the woundresponse, and it opens a new aspect of polyamines in plant biology.In contrast to animals, plants are unable to mobilize specialized cells devoted to wound healing after herbivore attach. Plant cells have evolved the capacity to activate defense responses that include wound healing and protection against further damage. Wound-activated repair/defense responses largely rely on the transcriptional activation of specific genes. These wound-inducible genes encode proteins implicated in activating wound signaling pathways, repairing damaged tissue, adjusting the metabolism for the production of toxins against the herbivore insect, and in the regulation of the internal nutritional demands (Leó n et al., 2001). In Arabidopsis, two different wound signaling pathways have been characterized (Titarenko et al., 1997). Upon wounding, expression of several genes is mediated by jasmonic acid (JA), whereas expression of others is independent of JA, but is activated by oligogalacturonides (Rojo et al., 1999). Very recently, a new mechanism, independent of JA or oligogalacturonides, has been reported to control the expression of the S-like RNase gene RNS1 (Taylor and Green, 1991) in systemically wounded tissue (LeBrasseur et al., 2002).The diamine putrescine and polyamines spermidine and spermine are small, ubiquitous compounds, positively charged at cytosolic pH, so they can interact with anionic macromolecules such as DNA, RNA, phospholipids, and proteins. Polyamines have been implicated in a variety of plant growth and developmental processes involving cell proliferation and differentiation, morphogenesis, dormancy and ge...