Javier Agustí

Strigolactone signaling is required for auxin-dependent stimulation of secondary growth in plants

Long distance cell-to-cell communication is critical for the development of multicellular organisms. In this respect, plants are especially demanding as they constantly integrate environmental inputs to adjust growth processes to different conditions. One example is thickening of shoots and roots, also designated as secondary growth. Secondary growth is mediated by the vascular cambium, a stem cell-like tissue whose cell-proliferating activity is regulated over a long distance by the plant hormone auxin. How auxin signaling is integrated at the level of cambium cells and how cambium activity is coordinated with other growth processes are largely unknown. Here, we provide physiological, genetic, and pharmacological evidence that strigolactones (SLs), a group of plant hormones recently described to be involved in the repression of shoot branching, positively regulate cambial activity and that this function is conserved among species. We show that SL signaling in the vascular cambium itself is sufficient for cambium stimulation and that it interacts strongly with the auxin signaling pathway. Our results provide a model of how auxin-based long-distance signaling is translated into cambium activity and suggest that SLs act as general modulators of plant growth forms linking the control of shoot branching with the thickening of stems and roots.

Strigolactones Suppress Adventitious Rooting in Arabidopsis and Pea

Adventitious root formation is essential for the propagation of many commercially important plant species and involves the formation of roots from nonroot tissues such as stems or leaves. Here, we demonstrate that the plant hormone strigolactone suppresses adventitious root formation in Arabidopsis (Arabidopsis thaliana) and pea (Pisum sativum). Strigolactone-deficient and response mutants of both species have enhanced adventitious rooting. CYCLIN B1 expression, an early marker for the initiation of adventitious root primordia in Arabidopsis, is enhanced in more axillary growth2 (max2), a strigolactone response mutant, suggesting that strigolactones restrain the number of adventitious roots by inhibiting the very first formative divisions of the founder cells. Strigolactones and cytokinins appear to act independently to suppress adventitious rooting, as cytokinin mutants are strigolactone responsive and strigolactone mutants are cytokinin responsive. In contrast, the interaction between the strigolactone and auxin signaling pathways in regulating adventitious rooting appears to be more complex. Strigolactone can at least partially revert the stimulatory effect of auxin on adventitious rooting, and auxin can further increase the number of adventitious roots in max mutants. We present a model depicting the interaction of strigolactones, cytokinins, and auxin in regulating adventitious root formation.

WOX4 Imparts Auxin Responsiveness to Cambium Cells in Arabidopsis

Extensive lateral growth of shoot and root axes is mediated mostly by the cambium, a meristematic tissue whose activity depends on the plant hormone auxin. This study reveals that the presence of the transcription factor WOX4 in cambium cells is essential for translating the accumulation of long distance–derived auxin into cell proliferation. Multipotent stem cell populations, the meristems, are fundamental for the indeterminate growth of plant bodies. One of these meristems, the cambium, is responsible for extended root and stem thickening. Strikingly, although the pivotal role of the plant hormone auxin in promoting cambium activity has been known for decades, the molecular basis of auxin responsiveness on the level of cambium cells has so far been elusive. Here, we reveal that auxin-dependent cambium stimulation requires the homeobox transcription factor WOX4. In Arabidopsis thaliana inflorescence stems, 1-N-naphthylphthalamic acid–induced auxin accumulation stimulates cambium activity in the wild type but not in wox4 mutants, although basal cambium activity is not abolished. This conclusion is confirmed by the analysis of cellular markers and genome-wide transcriptional profiling, which revealed only a small overlap between WOX4-dependent and cambium-specific genes. Furthermore, the receptor-like kinase PXY is required for a stable auxin-dependent increase in WOX4 mRNA abundance and the stimulation of cambium activity, suggesting a concerted role of PXY and WOX4 in auxin-dependent cambium stimulation. Thus, in spite of large anatomical differences, our findings uncover parallels between the regulation of lateral and apical plant meristems by demonstrating the requirement for a WOX family member for auxin-dependent regulation of lateral plant growth.

Analysis of secondary growth in the Arabidopsis shoot reveals a positive role of jasmonate signalling in cambium formation

After primary growth, most dicotyledonous plants undergo secondary growth. Secondary growth involves an increase in the diameter of shoots and roots through formation of secondary vascular tissue. A hallmark of secondary growth initiation in shoots of dicotyledonous plants is the initiation of meristematic activity between primary vascular bundles, i.e. in the interfascicular regions. This results in establishment of a cylindrical meristem, namely the vascular cambium. Surprisingly, despite its major implications for plant growth and the accumulation of biomass, the molecular regulation of secondary growth is only poorly understood. Here, we combine histological, molecular and genetic approaches to characterize interfascicular cambium initiation in the Arabidopsis thaliana inflorescence shoot. Using genome-wide transcriptional profiling, we show that stress-related and touch-inducible genes are up-regulated in stem regions where secondary growth takes place. Furthermore, we show that the products of COI1, MYC2, JAZ7 and the touch-inducible gene JAZ10, which are components of the JA signalling pathway, are cambium regulators. The positive effect of JA application on cambium activity confirmed a stimulatory role of JA in secondary growth, and suggests that JA signalling triggers cell divisions in this particular context.

Characterization of Transcriptome Remodeling during Cambium Formation Identifies MOL1 and RUL1 As Opposing Regulators of Secondary Growth

Cell-to-cell communication is crucial for the development of multicellular organisms, especially during the generation of new tissues and organs. Secondary growth—the lateral expansion of plant growth axes—is a highly dynamic process that depends on the activity of the cambium. The cambium is a stem cell–like tissue whose activity is responsible for wood production and, thus, for the establishment of extended shoot and root systems. Attempts to study cambium regulation at the molecular level have been hampered by the limitations of performing genetic analyses in trees and by the difficulty of accessing this tissue in model systems such as Arabidopsis thaliana. Here, we describe the roles of two receptor-like kinases, REDUCED IN LATERAL GROWTH1 (RUL1) and MORE LATERAL GROWTH1 (MOL1), as opposing regulators of cambium activity. Their identification was facilitated by a novel in vitro system in which cambium formation is induced in isolated Arabidopsis stem fragments. By combining this system with laser capture microdissection, we characterized transcriptome remodeling in a tissue- and stage-specific manner and identified series of genes induced during different phases of cambium formation. In summary, we provide a means for investigating cambium regulation in unprecedented depth and present two signaling components that control a process responsible for the accumulation of a large proportion of terrestrial biomass.

Analysis of 13000 unique Citrus clusters associated with fruit quality, production and salinity tolerance

BackgroundImprovement of Citrus, the most economically important fruit crop in the world, is extremely slow and inherently costly because of the long-term nature of tree breeding and an unusual combination of reproductive characteristics. Aside from disease resistance, major commercial traits in Citrus are improved fruit quality, higher yield and tolerance to environmental stresses, especially salinity.ResultsA normalized full length and 9 standard cDNA libraries were generated, representing particular treatments and tissues from selected varieties (Citrus clementina and C. sinensis) and rootstocks (C. reshni, and C. sinenis × Poncirus trifoliata) differing in fruit quality, resistance to abscission, and tolerance to salinity. The goal of this work was to provide a large expressed sequence tag (EST) collection enriched with transcripts related to these well appreciated agronomical traits. Towards this end, more than 54000 ESTs derived from these libraries were analyzed and annotated. Assembly of 52626 useful sequences generated 15664 putative transcription units distributed in 7120 contigs, and 8544 singletons. BLAST annotation produced significant hits for more than 80% of the hypothetical transcription units and suggested that 647 of these might be Citrus specific unigenes. The unigene set, composed of ~13000 putative different transcripts, including more than 5000 novel Citrus genes, was assigned with putative functions based on similarity, GO annotations and protein domainsConclusionComparative genomics with Arabidopsis revealed the presence of putative conserved orthologs and single copy genes in Citrus and also the occurrence of both gene duplication events and increased number of genes for specific pathways. In addition, phylogenetic analysis performed on the ammonium transporter family and glycosyl transferase family 20 suggested the existence of Citrus paralogs. Analysis of the Citrus gene space showed that the most important metabolic pathways known to affect fruit quality were represented in the unigene set. Overall, the similarity analyses indicated that the sequences of the genes belonging to these varieties and rootstocks were essentially identical, suggesting that the differential behaviour of these species cannot be attributed to major sequence divergences. This Citrus EST assembly contributes both crucial information to discover genes of agronomical interest and tools for genetic and genomic analyses, such as the development of new markers and microarrays.

Ethylene-induced differential gene expression during abscission of citrus leaves

The main objective of this work was to identify and classify genes involved in the process of leaf abscission in Clementina de Nules (Citrus clementina Hort. Ex Tan.). A 7 K unigene citrus cDNA microarray containing 12 K spots was used to characterize the transcriptome of the ethylene-induced abscission process in laminar abscission zone-enriched tissues and the petiole of debladed leaf explants. In these conditions, ethylene induced 100% leaf explant abscission in 72 h while, in air-treated samples, the abscission period started later and took 240 h. Gene expression monitored during the first 36 h of ethylene treatment showed that out of the 12 672 cDNA microarray probes, ethylene differentially induced 725 probes distributed as follows: 216 (29.8%) probes in the laminar abscission zone and 509 (70.2%) in the petiole. Functional MIPS classification and manual annotation of differentially expressed genes highlighted key processes regulating the activation and progress of the cell separation that brings about abscission. These included cell-wall modification, lipid transport, protein biosynthesis and degradation, and differential activation of signal transduction and transcription control pathways. Expression data associated with the petiole indicated the occurrence of a double defensive strategy mediated by the activation of a biochemical programme including scavenging ROS, defence and PR genes, and a physical response mostly based on lignin biosynthesis and deposition. This work identifies new genes probably involved in the onset and development of the leaf abscission process and suggests a different but co-ordinated and complementary role for the laminar abscission zone and the petiole during the process of abscission.

Comparative transcriptional survey between laser-microdissected cells from laminar abscission zone and petiolar cortical tissue during ethylene-promoted abscission in citrus leaves

BackgroundAbscission is the cell separation process by which plants are able to shed organs. It has a great impact on the yield of most crop plants. At the same time, the process itself also constitutes an excellent model to study cell separation processes, since it occurs in concrete areas known as abscission zones (AZs) which are composed of a specific cell type. However, molecular approaches are generally hampered by the limited area and cell number constituting the AZ. Therefore, detailed studies at the resolution of cell type are of great relevance in order to accurately describe the process and to identify potential candidate genes for biotechnological applications.ResultsEfficient protocols for the isolation of specific citrus cell types, namely laminar abscission zone (LAZ) and petiolar cortical (Pet) cells based on laser capture microdissection (LCM) and for RNA microextraction and amplification have been developed. A comparative transcriptome analysis between LAZ and Pet from citrus leaf explants subjected to an in-vitro 24 h ethylene treatment was performed utilising microarray hybridization and analysis. Our analyses of gene functional classes differentially represented in ethylene-treated LAZ revealed an activation program dominated by the expression of genes associated with protein synthesis, protein fate, cell type differentiation, development and transcription. The extensive repertoire of genes associated with cell wall biosynthesis and metabolism strongly suggests that LAZ layers activate both catabolic and anabolic wall modification pathways during the abscission program. In addition, over-representation of particular members of different transcription factor families suggests important roles for these genes in the differentiation of the effective cell separation layer within the many layers contained in the citrus LAZ. Preferential expression of stress-related and defensive genes in Pet reveals that this tissue is likely to be reprogrammed to prevent pathogen attacks and general abiotic stresses after organ shedding.ConclusionThe LCM-based data generated in this survey represent the most accurate description of the main biological processes and genes involved in organ abscission in citrus. This study provides novel molecular insight into ethylene-promoted leaf abscission and identifies new putative target genes for characterization and manipulation of organ abscission in citrus.

Tissue-specific transcriptome profiling of the citrus fruit epidermis and subepidermis using laser capture microdissection

Most studies of the biochemical and regulatory pathways that are associated with, and control, fruit expansion and ripening are based on homogenized bulk tissues, and do not take into consideration the multiplicity of different cell types from which the analytes, be they transcripts, proteins or metabolites, are extracted. Consequently, potentially valuable spatial information is lost and the lower abundance cellular components that are expressed only in certain cell types can be diluted below the level of detection. In this study, laser microdissection (LMD) was used to isolate epidermal and subepidermal cells from green, expanding Citrus clementina fruit and their transcriptomes were compared using a 20k citrus cDNA microarray and quantitative real-time PCR. The results show striking differences in gene expression profiles between the two cell types, revealing specific metabolic pathways that can be related to their respective organelle composition and cell wall specialization. Microscopy provided additional evidence of tissue specialization that could be associated with the transcript profiles with distinct differences in organelle and metabolite accumulation. Subepidermis predominant genes are primarily involved in photosynthesis- and energy-related processes, as well as cell wall biosynthesis and restructuring. By contrast, the most epidermis predominant genes are related to the biosynthesis of the cuticle, flavonoids, and defence responses. Furthermore, the epidermis transcript profile showed a high proportion of genes with no known function, supporting the original hypothesis that analysis at the tissue/cell specific levels can promote gene discovery and lead to a better understanding of the specialized contribution of each tissue to fruit physiology.

Going with the wind – Adaptive dynamics of plant secondary meristems

Highlights ► We discuss the role of secondary meristems in the adaptation of plant growth forms. ► We highlight the integration of environmental inputs into plant growth regulation. ► Branching and secondary growth shape the plant body. ► Plant’s phenotypic plasticity can be used to dissect the evolution of growth forms.