A genetic framework for proximal secondary vein branching in the Arabidopsis thaliana embryo.

Over time, plants have evolved flexible self-organizing patterning mechanisms to adapt tissue functionality for continuous organ growth. An example of this process is the multicellular organization of cells into a vascular network in foliar organs. An important, yet poorly understood component of this process is secondary vein branching, a mechanism employed to extend vascular tissues throughout the cotyledon surface. Here, we uncover two distinct branching mechanisms during embryogenesis by analyzing the discontinuous vein network of the double mutant cotyledon vascular pattern 2 (cvp2) cvp2-like 1 (cvl1). Similar to wild-type embryos, distal veins in cvp2 cvl1 embryos arise from the bifurcation of cell files contained in the midvein, whereas proximal branching is absent in this mutant. Restoration of this process can be achieved by increasing OCTOPUS dosage as well as by silencing RECEPTOR-LIKE PROTEIN KINASE 2 (RPK2) expression. Although RPK2-dependent rescue of cvp2 cvl1 is auxin- and CLE peptide-independent, distal branching involves polar auxin transport and follows a distinct regulatory mechanism. Our work defines a genetic network that confers plasticity to Arabidopsis embryos to spatially adapt vascular tissues to organ growth.

SOBIR1/EVR prevents precocious initiation of fiber differentiation during wood development through a mechanism involving BP and ERECTA.

In plants, secondary growth results in radial expansion of stems and roots, generating large amounts of biomass in the form of wood. Using genome-wide association studies (GWAS)-guided reverse genetics in Arabidopsis thaliana, we discovered SOBIR1/EVR, previously known to control plant immunoresponses and abscission, as a regulator of secondary growth. We present anatomical, genetic, and molecular evidence indicating that SOBIR1/EVR prevents the precocious differentiation of xylem fiber, a key cell type for wood development. SOBIR1/EVR acts through a mechanism that involves BREVIPEDICELLUS (BP) and ERECTA (ER), 2 proteins previously known to regulate xylem fiber development. We demonstrate that BP binds SOBIR1/EVR promoter and that SOBIR1/EVR expression is enhanced in bp mutants, suggesting a direct, negative regulation of BP over SOBIR1/EVR expression. We show that SOBIR1/EVR physically interacts with ER and that defects caused by the sobir1/evr mutation are aggravated by mutating ER, indicating that SOBIR1/EVR and ERECTA act together in the control of the precocious formation of xylem fiber development.

Gibberellins negatively modulate ovule number in plants.

Ovule formation is a complex developmental process in plants, with a strong impact on the production of seeds. Ovule primordia initiation is controlled by a gene network, including components of the signaling pathways of auxin, brassinosteroids and cytokinins. By contrast, gibberellins (GAs) and DELLA proteins, the negative regulators of GA signaling, have never been shown to be involved in ovule initiation. Here, we provide molecular and genetic evidence that points to DELLA proteins as novel players in the determination of ovule number in Arabidopsis and in species of agronomic interest, such as tomato and rapeseed, adding a new layer of complexity to this important developmental process. DELLA activity correlates positively with ovule number, acting as a positive factor for ovule initiation. In addition, ectopic expression of a dominant DELLA in the placenta is sufficient to increase ovule number. The role of DELLA proteins in ovule number does not appear to be related to auxin transport or signaling in the ovule primordia. Possible crosstalk between DELLA proteins and the molecular and hormonal network controlling ovule initiation is also discussed.

Fruit-dependent epigenetic regulation of flowering in Citrus.

In many perennial plants, seasonal flowering is primarily controlled by environmental conditions, but in certain polycarpic plants, environmental signals are locally gated by the presence of developing fruits initiated in the previous season through an unknown mechanism. Polycarpy is defined as the ability of plants to undergo several rounds of reproduction during their lifetime, alternating vegetative and reproductive meristems in the same individual. To understand how fruits regulate flowering in polycarpic plants, we focused on alternate bearing in Citrus trees that had been experimentally established as fully flowering or nonflowering. We found that the presence of the fruit causes epigenetic changes correlating with the induction of the CcMADS19 floral repressor, which prevents the activation of the floral promoter CiFT2 even in the presence of the floral inductive signals. By contrast, newly emerging shoots display an opposite epigenetic scenario associated with CcMADS19 repression, thereby allowing the activation of CiFT2 the following cold season.

Arabidopsis Heat Stress-Induced Proteins Are Enriched in Electrostatically Charged Amino Acids and Intrinsically Disordered Regions.

Comparison of the proteins of thermophilic, mesophilic, and psychrophilic prokaryotes has revealed several features characteristic to proteins adapted to high temperatures, which increase their thermostability. These characteristics include a profusion of disulfide bonds, salt bridges, hydrogen bonds, and hydrophobic interactions, and a depletion in intrinsically disordered regions. It is unclear, however, whether such differences can also be observed in eukaryotic proteins or when comparing proteins that are adapted to temperatures that are more subtly different. When an organism is exposed to high temperatures, a subset of its proteins is overexpressed (heat-induced proteins), whereas others are either repressed (heat-repressed proteins) or remain unaffected. Here, we determine the expression levels of all genes in the eukaryotic model system Arabidopsis thaliana at 22 and 37 °C, and compare both the amino acid compositions and levels of intrinsic disorder of heat-induced and heat-repressed proteins. We show that, compared to heat-repressed proteins, heat-induced proteins are enriched in electrostatically charged amino acids and depleted in polar amino acids, mirroring thermophile proteins. However, in contrast with thermophile proteins, heat-induced proteins are enriched in intrinsically disordered regions, and depleted in hydrophobic amino acids. Our results indicate that temperature adaptation at the level of amino acid composition and intrinsic disorder can be observed not only in proteins of thermophilic organisms, but also in eukaryotic heat-induced proteins; the underlying adaptation pathways, however, are similar but not the same.

Bud sprouting and floral induction and expression of FT in loquat [Eriobotrya japonica (Thunb.) Lindl.].

MAIN CONCLUSION: EjFT1 and EjFT2 genes were isolated and sequenced from leaves of loquat. EjFT1 is involved in bud sprouting and leaf development, and EjFT2 in floral bud induction. Loquat [Eriobotrya japonica (Thunb.) Lindl.] is an evergreen species belonging to the family Rosaceae, such as apple and pear, whose reproductive development, in contrast with these species, is a continuous process that is not interrupted by winter dormancy. Thus, the study of the mechanism of flowering in loquat has the potential to uncover the environmental and genetic networks that trigger flowering more accurately, contributing for a better understanding of the Rosaceae floral process. As a first step toward understanding the molecular mechanisms controlling flowering, extensive defoliation and defruiting assays, together with molecular studies of the key FLOWERING LOCUS T (FT) gene, were carried out. FT exhibited two peaks of expression in leaves, the first one in early to mid-May, the second one in mid-June. Two FT genes, EjFT1 and EjFT2, were isolated and sequenced and studied their expression. Expression of EjFT1 and EjFT2 peaks in mid-May, at bud sprouting. EjFT2 expression peaks again in mid-June, coinciding with the floral bud inductive period. Thus, when all leaves of the tree were continuously removed from early to late May vegetative apex differentiated into panicle, but when defoliation was performed from early to late June apex did not differentiate. On the other hand, fruit removal advanced EjFT1 expression in old leaves and the sooner the fruit detached, the sooner the bud sprouted. Accordingly, results strongly suggest that EjFT1 might be related to bud sprouting and leaf development, while EjFT2 might be involved in floral bud induction. An integrative model for FT functions in loquat is discussed.

α2-COP is involved in early secretory traffic in Arabidopsis and is required for plant growth.

COP (coat protein) I-coated vesicles mediate intra-Golgi transport and retrograde transport from the Golgi to the endoplasmic reticulum. These vesicles form through the action of the small GTPase ADP-ribosylation factor 1 (ARF1) and the COPI heptameric protein complex (coatomer), which consists of seven subunits (α-, ß-, ß'-, γ-, δ-, ε- and ζ-COP). In contrast to mammals and yeast, several isoforms for coatomer subunits, with the exception of γ and δ, have been identified in Arabidopsis. To understand the role of COPI proteins in plant biology, we have identified and characterized a loss-of-function mutant of α2-COP, an Arabidopsis α-COP isoform. The α2-cop mutant displayed defects in plant growth, including small rosettes, stems and roots and mislocalization of p24δ5, a protein of the p24 family containing a C-terminal dilysine motif involved in COPI binding. The α2-cop mutant also exhibited abnormal morphology of the Golgi apparatus. Global expression analysis of the α2-cop mutant revealed altered expression of plant cell wall-associated genes. In addition, a strong upregulation of SEC31A, which encodes a subunit of the COPII coat, was observed in the α2-cop mutant; this also occurs in a mutant of a gene upstream of COPI assembly, GNL1, which encodes an ARF-guanine nucleotide exchange factor (GEF). These findings suggest that loss of α2-COP affects the expression of secretory pathway genes.

Arabidopsis PYR/PYL/RCAR receptors play a major role in quantitative regulation of stomatal aperture and transcriptional response to abscisic acid.

Abscisic acid (ABA) is a key hormone for plant growth, development, and stress adaptation. Perception of ABA through four types of receptors has been reported. We show here that impairment of ABA perception through the PYRABACTIN RESISTANCE1 (PYR1)/PYR1-LIKE (PYL)/REGULATORY COMPONENTS OF ABA RECEPTORS (RCAR) branch reduces vegetative growth and seed production and leads to a severe open stomata and ABA-insensitive phenotype, even though other branches for ABA perception remain functional. An Arabidopsis thaliana sextuple mutant impaired in six PYR/PYL receptors, namely PYR1, PYL1, PYL2, PYL4, PYL5, and PYL8, was able to germinate and grow even on 100 µM ABA. Whole-rosette stomatal conductance (Gst) measurements revealed that leaf transpiration in the sextuple pyr/pyl mutant was higher than in the ABA-deficient aba3-1 or ABA-insensitive snrk2.6 mutants. The gradually increasing Gst values of plants lacking three, four, five, and six PYR/PYLs indicate quantitative regulation of stomatal aperture by this family of receptors. The sextuple mutant lacked ABA-mediated activation of SnRK2s, and ABA-responsive gene expression was dramatically impaired as was reported in snrk2.2/2.3/2.6. In summary, these results show that ABA perception by PYR/PYLs plays a major role in regulation of seed germination and establishment, basal ABA signaling required for vegetative and reproductive growth, stomatal aperture, and transcriptional response to the hormone.

Thermospermine levels are controlled by an auxin-dependent feedback loop mechanism in Populus xylem.

Polyamines are small polycationic amines that are widespread in living organisms. Thermospermine, synthesized by thermospermine synthase ACAULIS5 (ACL5), was recently shown to be an endogenous plant polyamine. Thermospermine is critical for proper vascular development and xylem cell specification, but it is not known how thermospermine homeostasis is controlled in the xylem. We present data in the Populus model system supporting the existence of a negative feedback control of thermospermine levels in stem xylem tissues, the main site of thermospermine biosynthesis. While over-expression of the ACL5 homologue in Populus, POPACAULIS5, resulted in strong up-regulation of ACL5 expression and thermospermine accumulation in leaves, the corresponding levels in the secondary xylem tissues of the stem were similar or lower than those in the wild-type. POPACAULIS5 over-expression had a negative effect on accumulation of indole-3-acetic acid, while exogenous auxin had a positive effect on POPACAULIS5 expression, thus promoting thermospermine accumulation. Further, over-expression of POPACAULIS5 negatively affected expression of the class III homeodomain leucine zipper (HD-Zip III) transcription factor gene PttHB8, a homologue of AtHB8, while up-regulation of PttHB8 positively affected POPACAULIS5 expression. These results indicate that excessive accumulation of thermospermine is prevented by a negative feedback control of POPACAULIS5 transcript levels through suppression of indole-3-acetic acid levels, and that PttHB8 is involved in the control of POPACAULIS5 expression. We propose that this negative feedback loop functions to maintain steady-state levels of thermospermine, which is required for proper xylem development, and that it is dependent on the presence of high concentrations of endogenous indole-3-acetic acid, such as those present in the secondary xylem tissues.

Arabidopsis copper transport protein COPT2 participates in the cross talk between iron deficiency responses and low-phosphate signaling.

Copper and iron are essential micronutrients for most living organisms because they participate as cofactors in biological processes, including respiration, photosynthesis, and oxidative stress protection. In many eukaryotic organisms, including yeast (Saccharomyces cerevisiae) and mammals, copper and iron homeostases are highly interconnected; yet, such interdependence is not well established in higher plants. Here, we propose that COPT2, a high-affinity copper transport protein, functions under copper and iron deficiencies in Arabidopsis (Arabidopsis thaliana). COPT2 is a plasma membrane protein that functions in copper acquisition and distribution. Characterization of the COPT2 expression pattern indicates a synergic response to copper and iron limitation in roots. We characterized a knockout of COPT2, copt2-1, that leads to increased resistance to simultaneous copper and iron deficiencies, measured as reduced leaf chlorosis and improved maintenance of the photosynthetic apparatus. We propose that COPT2 could play a dual role under iron deficiency. First, COPT2 participates in the attenuation of copper deficiency responses driven by iron limitation, possibly to minimize further iron consumption. Second, global expression analyses of copt2-1 versus wild-type Arabidopsis plants indicate that low-phosphate responses increase in the mutant. These results open up new biotechnological approaches to fight iron deficiency in crops.

Molecular programme of senescence in dry and fleshy fruits.

Fruits of angiosperms can be divided into dry and fleshy fruits, depending on their dispersal strategies. Despite their apparently different developmental programmes, researchers have attempted to compare dry and fleshy fruits to establish analogies of the distinct biochemical and physiological processes that occur. But what are the common and specific phenomena in both biological strategies? Is valve dehiscence and senescence of dry fruits comparable to final ripening of fleshy fruits, when seeds become mature and fruits are competent for seed dispersal, or to over-ripening when advanced senescence occurs? We briefly review current knowledge on dry and fleshy fruit development, which has been extensively reported recently, and is the topic of this special issue. We compare the processes taking place in Arabidopsis (dry) and tomato (fleshy) fruit during final development steps using transcriptome data to establish possible analogies. Interestingly, the transcriptomic programme of Arabidopsis silique shares little similarity in gene number to tomato fruit ripening or over-ripening. In contrast, the biological processes carried out by these common genes from ripening and over-ripening programmes are similar, as most biological processes are shared during both programmes. On the other hand, several biological terms are specific of Arabidopsis and tomato ripening, including senescence, but little or no specific processes occur during Arabidopsis and tomato over-ripening. These suggest a closer analogy between silique senescence and ripening than over-ripening, but a major common biological programme between Arabidopsis silique senescence and the last steps of tomato development, irrespective of its distinction between ripening and over-ripening.

A plant spermine oxidase/dehydrogenase regulated by the proteasome and polyamines.

Polyamine oxidases (PAOs) are flavin-dependent enzymes involved in polyamine catabolism. In Arabidopsis five PAO genes (AtPAO1-AtPAO5) have been identified which present some common characteristics, but also important differences in primary structure, substrate specificity, subcellular localization, and tissue-specific expression pattern, differences which may suggest distinct physiological roles. In the present work, AtPAO5, the only so far uncharacterized AtPAO which is specifically expressed in the vascular system, was partially purified from 35S::AtPAO5-6His Arabidopsis transgenic plants and biochemically characterized. Data presented here allow AtPAO5 to be classified as a spermine dehydrogenase. It is also shown that AtPAO5 oxidizes the polyamines spermine, thermospermine, and N(1)-acetylspermine, the latter being the best in vitro substrate of the recombinant enzyme. AtPAO5 also oxidizes these polyamines in vivo, as was evidenced by analysis of polyamine levels in the 35S::AtPAO5-6His Arabidopsis transgenic plants, as well as in a loss-of-function atpao5 mutant. Furthermore, subcellular localization studies indicate that AtPAO5 is a cytosolic protein undergoing proteasomal control. Positive regulation of AtPAO5 expression by polyamines at the transcriptional and post-transcriptional level is also shown. These data provide new insights into the catalytic properties of the PAO gene family and the complex regulatory network controlling polyamine metabolism.

Quantification of Tracheary Elements Types in Mature Hypocotyl of Arabidopsis thaliana.

Secondary growth is a highly relevant process for dicot and gymnosperm species development. The process relies on vascular tissue proliferation and culminates with the thickening of stems, roots, and hypocotyls. The formation of tracheary elements is a critical step during this process. Among such tracheary elements, four different cell types are distinguished depending on their secondary cell wall pattern, which is exclusive for each tracheary cell type. Here we describe a method to isolate, dye, and recognize each of these tracheary cell types. The method is optimized to be performed in the Arabidopsis thaliana hypocotyl. This is because, in this species, the hypocotyl is the organ undergoing the largest proportion of secondary growth. Results allow for determining the relative amounts of each of the tracheary cell types.

Signalling mechanisms and agricultural applications of (Z)-3-hexenyl butyrate-mediated stomatal closure.

Biotic and abiotic stresses can severely limit crop productivity. In response to drought, plants close stomata to prevent water loss. Furthermore, stomata are the main entry point for several pathogens. Therefore, the development of natural products to control stomata closure can be considered a sustainable strategy to cope with stresses in agriculture. Plants respond to different stresses by releasing volatile organic compounds. Green leaf volatiles, which are commonly produced across different plant species after tissue damage, comprise an important group within volatile organic compounds. Among them, (Z)-3-hexenyl butyrate (HB) was described as a natural inducer of stomatal closure, playing an important role in stomatal immunity, although its mechanism of action is still unknown. Through different genetic, pharmacological, and biochemical approaches, we here uncover that HB perception initiates various defence signalling events, such as activation of Ca2+ permeable channels, mitogen-activated protein kinases, and production of Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase-mediated reactive oxygen species. Furthermore, HB-mediated stomata closure was found to be independent of abscisic acid biosynthesis and signalling. Additionally, exogenous treatments with HB alleviate water stress and improve fruit productivity in tomato plants. The efficacy of HB was also tested under open field conditions, leading to enhanced resistance against Phytophthora spp. and Pseudomonas syringae infection in potato and tomato plants, respectively. Taken together, our results provide insights into the HB signalling transduction pathway, confirming its role in stomatal closure and plant immune system activation, and propose HB as a new phytoprotectant for the sustainable control of biotic and abiotic stresses in agriculture.

Quantitation of biogenic tetraamines in Arabidopsis thaliana.

Classical methods for the determination of polyamines in Arabidopsis, based on high-performance liquid chromatography or thin-layer chromatography, do not distinguish between aliphatic tetraamines or do not allow accurate quantitation, respectively. Here we describe a gas chromatography-mass spectrometry method that allows separation and simple quantitation of spermine and thermospermine. The reliability of the protocol has been confirmed using extracts of Arabidopsis mutants impaired in tetraamine biosynthesis. The levels of thermospermine are around 6-fold lower than those of spermine in Arabidopsis seedlings. Interestingly, hormonal effects on polyamine levels are not always correlated with those of transcripts of their biosynthetic enzymes.

Deregulated High Affinity Copper Transport Alters Iron Homeostasis in Arabidopsis.

The present work describes the effects on iron homeostasis when copper transport was deregulated in Arabidopsis thaliana by overexpressing high affinity copper transporters COPT1 and COPT3 (COPTOE ). A genome-wide analysis conducted on COPT1OE plants, highlighted that iron homeostasis gene expression was affected under both copper deficiency and excess. Among the altered genes were those encoding the iron uptake machinery and their transcriptional regulators. Subsequently, COPTOE seedlings contained less iron and were more sensitive than controls to iron deficiency. The deregulation of copper (I) uptake hindered the transcriptional activation of the subgroup Ib of basic helix-loop-helix (bHLH-Ib) factors under copper deficiency. Oppositely, copper excess inhibited the expression of the master regulator FIT but activated bHLH-Ib expression in COPTOE plants, in both cases leading to the lack of an adequate iron uptake response. As copper increased in the media, iron (III) was accumulated in roots, and the ratio iron (III)/iron (II) was increased in COPTOE plants. Thus, iron (III) overloading in COPTOE roots inhibited local iron deficiency responses, aimed to metal uptake from soil, leading to a general lower iron content in the COPTOE seedlings. These results emphasized the importance of appropriate spatiotemporal copper uptake for iron homeostasis under non-optimal copper supply. The understanding of the role of copper uptake in iron metabolism could be applied for increasing crops resistance to iron deficiency.

Evolutionary diversification in polyamine biosynthesis.

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.

A bHLH-Based Feedback Loop Restricts Vascular Cell Proliferation in Plants.

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.

Role of polyamines in plant vascular development.

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.

ACAULIS5 controls Arabidopsis xylem specification through the prevention of premature cell death.

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.