Genetic Diversity and Population Structure Assessed Using Microsatellite (SSR) Markers from Relict Populations of Nuphar pumila (Nymphaeaceae).

The genus Nuphar (Nymphaeaceae) comprises aquatic plant species inhabiting freshwater bodies of the Northern hemisphere temperate regions known as yellow water-lilies. Nuphar lutea and N. pumila are the only representatives in the European continent and present different ecologies: the former is a widespread generalist, while the latter is restricted to northern latitudes or high-altitudes due to its requirements for colder and oligotrophic waters. The Central Europe mountainous areas, the Massif Central (France) and the Cantabrian Mountains (north Iberian Peninsula) harbor relict isolated N. pumila populations endangered by eutrophication and hybridization with N. lutea. We aim to detect hybridization processes in the Massif Central and Cantabrian Mountains populations and compare the genetic diversity of N. pumila in the relict populations of Central Europe by using microsatellite (SSR) markers. No evidence of hybridization was found in the Iberian population, whereas the admixture between N. pumila and N. lutea in the Massif Central populations could be due to hybridization or ancient introgression. Our current knowledge would benefit from genetic diversity studies focusing on both species throughout their distributional range. The Iberian and Massif Central N. pumila populations were genetically distinct, representing two different clusters from other relict populations, with low genetic diversity and a genetic boundary within Central Europe.

In Vitro Plant Regeneration in Conifers: The Role of WOX and KNOX Gene Families.

Conifers are a group of woody plants with an enormous economic and ecological importance. Breeding programs are necessary to select superior varieties for planting, but they have many limitations due to the biological characteristics of conifers. Somatic embryogenesis (SE) and de novo organogenesis (DNO) from in vitro cultured tissues are two ways of plant mass propagation that help to overcome this problem. Although both processes are difficult to achieve in conifers, they offer advantages like a great efficiency, the possibilities to cryopreserve the embryogenic lines, and the ability of multiplying adult trees (the main bottleneck in conifer cloning) through DNO. Moreover, SE and DNO represent appropriate experimental systems to study the molecular bases of developmental processes in conifers such as embryogenesis and shoot apical meristem (SAM) establishment. Some of the key genes regulating these processes belong to the WOX and KNOX homeobox gene families, whose function has been widely described in Arabidopsis thaliana. The sequences and roles of these genes in conifers are similar to those found in angiosperms, but some particularities exist, like the presence of WOXX, a gene that putatively participates in the establishment of SAM in somatic embryos and plantlets of Pinus pinaster.

Modulation of plant root growth by nitrogen source-defined regulation of polar auxin transport.

Availability of the essential macronutrient nitrogen in soil plays a critical role in plant growth, development, and impacts agricultural productivity. Plants have evolved different strategies for sensing and responding to heterogeneous nitrogen distribution. Modulation of root system architecture, including primary root growth and branching, is among the most essential plant adaptions to ensure adequate nitrogen acquisition. However, the immediate molecular pathways coordinating the adjustment of root growth in response to distinct nitrogen sources, such as nitrate or ammonium, are poorly understood. Here, we show that growth as manifested by cell division and elongation is synchronized by coordinated auxin flux between two adjacent outer tissue layers of the root. This coordination is achieved by nitrate-dependent dephosphorylation of the PIN2 auxin efflux carrier at a previously uncharacterized phosphorylation site, leading to subsequent PIN2 lateralization and thereby regulating auxin flow between adjacent tissues. A dynamic computer model based on our experimental data successfully recapitulates experimental observations. Our study provides mechanistic insights broadening our understanding of root growth mechanisms in dynamic environments.

The Arabidopsis NRT1.1 transceptor coordinately controls auxin biosynthesis and transport to regulate root branching in response to nitrate.

In agricultural systems, nitrate is the main source of nitrogen available for plants. Besides its role as a nutrient, nitrate has been shown to act as a signal molecule in plant growth, development, and stress responses. In Arabidopsis, the NRT1.1 nitrate transceptor represses lateral root (LR) development at low nitrate availability by promoting auxin basipetal transport out of the LR primordia (LRPs). Here we show that NRT1.1 acts as a negative regulator of the TAR2 auxin biosynthetic gene in the root stele. This is expected to repress local auxin biosynthesis and thus to reduce acropetal auxin supply to the LRPs. Moreover, NRT1.1 also negatively affects expression of the LAX3 auxin influx carrier, thus preventing the cell wall remodeling required for overlying tissue separation during LRP emergence. NRT1.1-mediated repression of both TAR2 and LAX3 is suppressed at high nitrate availability, resulting in nitrate induction of the TAR2 and LAX3 expression that is required for optimal stimulation of LR development by nitrate. Altogether, our results indicate that the NRT1.1 transceptor coordinately controls several crucial auxin-associated processes required for LRP development, and as a consequence that NRT1.1 plays a much more integrated role than previously expected in regulating the nitrate response of root system architecture.

SYNERGISTIC ON AUXIN AND CYTOKININ 1 positively regulates growth and attenuates soil pathogen resistance.

Plants as non-mobile organisms constantly integrate varying environmental signals to flexibly adapt their growth and development. Local fluctuations in water and nutrient availability, sudden changes in temperature or other abiotic and biotic stresses can trigger changes in the growth of plant organs. Multiple mutually interconnected hormonal signaling cascades act as essential endogenous translators of these exogenous signals in the adaptive responses of plants. Although the molecular backbones of hormone transduction pathways have been identified, the mechanisms underlying their interactions are largely unknown. Here, using genome wide transcriptome profiling we identify an auxin and cytokinin cross-talk component; SYNERGISTIC ON AUXIN AND CYTOKININ 1 (SYAC1), whose expression in roots is strictly dependent on both of these hormonal pathways. We show that SYAC1 is a regulator of secretory pathway, whose enhanced activity interferes with deposition of cell wall components and can fine-tune organ growth and sensitivity to soil pathogens.

Hormonal and gene dynamics in de novo shoot meristem formation during adventitious caulogenesis in cotyledons of Pinus pinea.

KEY MESSAGE: Several members of WOX and KNOX gene families and several plant growth regulators, basically cytokinins and auxins, play a key role during adventitious caulogenesis in the conifer Pinus pinea. Similar to Arabidopsis thaliana, Pinus pinea shoot organogenesis is a multistep process. However, there are key differences between both species, which may alter the underlying physiological and genetic programs. It is unknown if the genic expression models during angiosperm development may be applicable to conifers. In this work, an analysis of the endogenous content of different plant growth regulators and the expression of genes putatively involved in adventitious caulogenesis in P. pinea cotyledons was conducted. A multivariate analysis of both datasets was also realized through partial least squares regression and principal component analysis to obtain an integral vision of the mechanisms involved in caulogenesis in P. pinea. Analyses show that cotyledons cultured in the presence of benzyladenine during long times (2-6 days) cluster separately from the rest of the samples, suggesting that the benzyladenine increase observed during the first hours of culture is sufficient to trigger the caulogenic response through the activation of specific developmental programs. In particular, the most relevant factors involved in this process are the cytokinins trans-zeatin, dihydrozeatin, trans-zeatin riboside and isopentenyl adenosine; the auxin indoleacetic acid; and the genes PpWUS, PpWOX5, PpKN2, PpKN3 and PipiRR1. WUS is functional in pines and has an important role in caulogenesis. Interestingly, WOX5 also seems to participate in the process, although its specific role has not been determined.

CUP-SHAPED COTYLEDON1 (CUC1) and CUC2 regulate cytokinin homeostasis to determine ovule number in Arabidopsis.

Seeds derive from ovules upon fertilization and therefore the total number of ovules determines the final seed yield, a fundamental trait in crop plants. Among the factors that co-ordinate the process of ovule formation, the transcription factors CUP-SHAPED COTYLEDON 1 (CUC1) and CUC2 and the hormone cytokinin (CK) have a particularly prominent role. Indeed, the absence of both CUC1 and CUC2 causes a severe reduction in ovule number, a phenotype that can be rescued by CK treatment. In this study, we combined CK quantification with an integrative genome-wide target identification approach to select Arabidopsis genes regulated by CUCs that are also involved in CK metabolism. We focused our attention on the functional characterization of UDP-GLUCOSYL TRANSFERASE 85A3 (UGT85A3) and UGT73C1, which are up-regulated in the absence of CUC1 and CUC2 and encode enzymes able to catalyse CK inactivation by O-glucosylation. Our results demonstrate a role for these UGTs as a link between CUCs and CK homeostasis, and highlight the importance of CUCs and CKs in the determination of seed yield.

Light Controls Cytokinin Signaling via Transcriptional Regulation of Constitutively Active Sensor Histidine Kinase CKI1.

In plants, the multistep phosphorelay (MSP) pathway mediates a range of regulatory processes, including those activated by cytokinins. The cross talk between cytokinin response and light has been known for a long time. However, the molecular mechanism underlying the interaction between light and cytokinin signaling remains elusive. In the screen for upstream regulators we identified a LONG PALE HYPOCOTYL (LPH) gene whose activity is indispensable for spatiotemporally correct expression of CYTOKININ INDEPENDENT1 (CKI1), encoding the constitutively active sensor His kinase that activates MSP signaling. lph is a new allele of HEME OXYGENASE1 (HY1) that encodes the key protein in the biosynthesis of phytochromobilin, a cofactor of photoconvertible phytochromes. Our analysis confirmed the light-dependent regulation of the CKI1 expression pattern. We show that CKI1 expression is under the control of phytochrome A (phyA), functioning as a dual (both positive and negative) regulator of CKI1 expression, presumably via the phyA-regulated transcription factors (TF) PHYTOCHROME INTERACTING FACTOR3 and CIRCADIAN CLOCK ASSOCIATED1. Changes in CKI1 expression observed in lph/hy1-7 and phy mutants correlate with misregulation of MSP signaling, changed cytokinin sensitivity, and developmental aberrations that were previously shown to be associated with cytokinin and/or CKI1 action. Besides that, we demonstrate a novel role of phyA-dependent CKI1 expression in the hypocotyl elongation and hook development during skotomorphogenesis. Based on these results, we propose that the light-dependent regulation of CKI1 provides a plausible mechanistic link underlying the well-known interaction between light- and cytokinin-controlled plant development.

Nitrate Controls Root Development through Posttranscriptional Regulation of the NRT1.1/NPF6.3 Transporter/Sensor.

Plants are able to modulate root growth and development to optimize their nitrogen nutrition. In Arabidopsis (Arabidopsis thaliana), the adaptive root response to nitrate (NO3-) depends on the NRT1.1/NPF6.3 transporter/sensor. NRT1.1 represses emergence of lateral root primordia (LRPs) at low concentration or absence of NO3- through its auxin transport activity that lowers auxin accumulation in LR. However, these functional data strongly contrast with the known transcriptional regulation of NRT1.1, which is markedly repressed in LRPs in the absence of NO3- To explain this discrepancy, we investigated in detail the spatiotemporal expression pattern of the NRT1.1 protein during LRP development and combined local transcript analysis with the use of transgenic lines expressing tagged NRT1.1 proteins. Our results show that although NO3- stimulates NRT1.1 transcription and probably mRNA stability both in primary root tissues and in LRPs, it acts differentially on protein accumulation, depending on the tissues considered with stimulation in cortex and epidermis of the primary root and a strong repression in LRPs and to a lower extent at the primary root tip. This demonstrates that NRT1.1 is strongly regulated at the posttranscriptional level by tissue-specific mechanisms. These mechanisms are crucial for controlling the large palette of adaptive responses to NO3- mediated by NRT1.1 as they ensure that the protein is present in the proper tissue under the specific conditions where it plays a signaling role in this particular tissue.

Cytokinin response factors regulate PIN-FORMED auxin transporters.

Auxin and cytokinin are key endogenous regulators of plant development. Although cytokinin-mediated modulation of auxin distribution is a developmentally crucial hormonal interaction, its molecular basis is largely unknown. Here we show a direct regulatory link between cytokinin signalling and the auxin transport machinery uncovering a mechanistic framework for cytokinin-auxin cross-talk. We show that the CYTOKININ RESPONSE FACTORS (CRFs), transcription factors downstream of cytokinin perception, transcriptionally control genes encoding PIN-FORMED (PIN) auxin transporters at a specific PIN CYTOKININ RESPONSE ELEMENT (PCRE) domain. Removal of this cis-regulatory element effectively uncouples PIN transcription from the CRF-mediated cytokinin regulation and attenuates plant cytokinin sensitivity. We propose that CRFs represent a missing cross-talk component that fine-tunes auxin transport capacity downstream of cytokinin signalling to control plant development.

Physiological and biochemical responses to severe drought stress of nine Eucalyptus globulus clones: a multivariate approach.

Seasonal drought, typical of temperate and Mediterranean environments, creates problems in establishing plantations and affects development and yield, and it has been widely studied in numerous species. Forestry fast-growing species such as Eucalyptus spp. are an important resource in such environments, selected clones being generally used for production purposes in plantations in these areas. However, use of mono-specific plantations increases risk of plant loss due to abiotic stresses, making it essential to understand differences in an individual clone's physiological responses to drought stress. In order to study clonal differences in drought responses, nine Eucalyptus globulus (Labill.) clones (C14, C46, C97, C120, C222, C371, C405, C491 and C601) were gradually subjected to severe drought stress (<14% of field capacity). A total of 31 parameters, physiological (e.g., photosynthesis, gas exchange), biochemical (e.g., chlorophyll content) and hormonal (abscisic acid [ABA] content), were analysed by classic and multivariate techniques. Relationships between parameters were established, allowing related measurements to be grouped into functional units (pigment, growth, water and ABA). Differences in these units showed that there were two distinct groups of E. globulus clones on the basis of their different strategies when faced with drought stress. The C14 group (C14, C120, C405, C491 and C601) clones behave as water savers, maintaining high water content and showing high stomatal adjustment, and reducing their aerial growth to a great extent. The C46 group (C46, C97, C222 and C371) clones behave as water spenders, reducing their water content drastically and presenting osmotic adjustment. The latter maintains the highest growth rate under the conditions tested. The method presented here can be used to identify appropriate E. globulus clones for drought environments, facilitating the selection of material for production and repopulation environments.

An integrative model of the control of ovule primordia formation.

Upon hormonal signaling, ovules develop as lateral organs from the placenta. Ovule numbers ultimately determine the number of seeds that develop, and thereby contribute to the final seed yield in crop plants. We demonstrate here that CUP-SHAPED COTYLEDON 1 (CUC1), CUC2 and AINTEGUMENTA (ANT) have additive effects on ovule primordia formation. We show that expression of the CUC1 and CUC2 genes is required to redundantly regulate expression of PINFORMED1 (PIN1), which in turn is required for ovule primordia formation. Furthermore, our results suggest that the auxin response factor MONOPTEROS (MP/ARF5) may directly bind ANT, CUC1 and CUC2 and promote their transcription. Based on our findings, we propose an integrative model to describe the molecular mechanisms of the early stages of ovule development.

Systems approaches to study root architecture dynamics.

The plant root system is essential for providing anchorage to the soil, supplying minerals and water, and synthesizing metabolites. It is a dynamic organ modulated by external cues such as environmental signals, water and nutrients availability, salinity and others. Lateral roots (LRs) are initiated from the primary root post-embryonically, after which they progress through discrete developmental stages which can be independently controlled, providing a high level of plasticity during root system formation. Within this review, main contributions are presented, from the classical forward genetic screens to the more recent high-throughput approaches, combined with computer model predictions, dissecting how LRs and thereby root system architecture is established and developed.

Spatiotemporal regulation of lateral root organogenesis in Arabidopsis by cytokinin.

The architecture of a plant's root system, established postembryonically, results from both coordinated root growth and lateral root branching. The plant hormones auxin and cytokinin are central endogenous signaling molecules that regulate lateral root organogenesis positively and negatively, respectively. Tight control and mutual balance of their antagonistic activities are particularly important during the early phases of lateral root organogenesis to ensure continuous lateral root initiation (LRI) and proper development of lateral root primordia (LRP). Here, we show that the early phases of lateral root organogenesis, including priming and initiation, take place in root zones with a repressed cytokinin response. Accordingly, ectopic overproduction of cytokinin in the root basal meristem most efficiently inhibits LRI. Enhanced cytokinin responses in pericycle cells between existing LRP might restrict LRI near existing LRP and, when compromised, ectopic LRI occurs. Furthermore, our results demonstrate that young LRP are more sensitive to perturbations in the cytokinin activity than are developmentally more advanced primordia. We hypothesize that the effect of cytokinin on the development of primordia possibly depends on the robustness and stability of the auxin gradient.

Rapid responses of C14 clone of Eucalyptus globulus to root drought stress: Time-course of hormonal and physiological signaling.

The responses of juvenile plants of forest crops to drought stress are a key stage in the survival of forest populations. In this work, a suitable experimental system to study the early drought resistance mechanisms and signaling in a drought-tolerant clone (C14) of Eucalyptus globulus Labill is proposed. This system, using hydroponic culture and an osmotic agent, polyethylene glycol 8000, was demonstrated to induce severe stress in the root area, affecting the responses of the plantlets at the aerial level. These responses were very fast, beginning only 3h after the induction of stress, and the results highlight the roles of xylematic abscisic acid (ABA) and pH changes over other signals, such as cytokinins, as early chemical signals in rapid water stress. The relationship between these chemical factors, ABA and pH, and the physiological and water parameters observed were significant, supporting their proposed principal role. This work aids our understanding of underlying responses to hydrological limitations of forest crops, and provides valuable information for further physiological and molecular studies of water stress in this and other tree species.

Benzyladenine metabolism and temporal competence of Pinus pinea cotyledons to form buds in vitro.

Germination negatively affects adventitious shoot formation induced by cytokinins in pine cotyledons. To investigate the causes of this decrease in the organogenic response, uptake and metabolism of benzyladenine (BA) were studied in stone pine cotyledons (Pinus pinea) isolated from in vitro germinating embryos and cultured in bud induction medium. As embryos grew, cotyledons showed a progressive decrease in the amount of BA taken up from the medium. BA was barely metabolized; however, a BA metabolite previously undescribed in conifers was found. It was identified as a glucoside of the BA riboside, a type of metabolite recently described in other gymnosperms. Data revealed that differences in the organogenic capacity of P. pinea cotyledons associated with embryo germination are related primarily to their ability to absorb BA from the bud induction medium.

Caulogenic induction in cotyledons of stone pine (Pinus pinea): relationship between organogenic response and benzyladenine trends in selected families.

Adventitious bud formation in stone pine cotyledons cultured in the presence of benzyladenine (BA) has been proposed as a model for the study of in vitro shoot organogenesis in conifers. This is because of its advantageous characteristics including the requirement of only one plant growth regulator (BA), the synchronous fashion of its induction, and the homogeneity and low degree of differentiation of cotyledons. Although optimal culture conditions have been developed and are currently in use, we still lack data for BA dynamics in cotyledons cultured under these conditions, and the morphological description of the early induction stages has not, until now, been approached from a histological perspective. Consequently, this is the focus of the present report. Additionally, we examined uptake and metabolism of BA in cotyledons from two selected families, previously characterized by, and selected for, the difference in the magnitude of their organogenic response. Media transfer experiments established that cotyledons should be in contact with 44.4 microM BA for at least 6h to obtain any caulogenic response (minimum shoot-induction period). Histological observations, carried out here for the first time in this species, determined that meristemoid structures had already begun to appear in explants within 12 h of culture. Moreover, results from the BA uptake and metabolism experiments indicated that the point at which explants reached the maximum concentration of active forms of BA (276.60 microM at 6 h) and the onset of the determination phase of shoot organogenesis were directly related. A direct relationship was also observed between the intensity of the caulogenic response in cotyledons from families 36 and 61 and the endogenous concentration of BA and its riboside at the start of the induction phase. Hence, family 36, characterized by its higher bud production, reached concentrations of 251.56 microM, while family 61, selected for its low bud-producing trait, only attained 175.80 microM. Finally, a correlation was observed between 6-benzylamino-9-[O-glucopyranosyl-(1-->3)ribofuranosyl]-purine values and the magnitude of the shoot organogenesis response.