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Quantifying healthy and degraded inner tissues in plants is of great interest in agronomy, for example, to assess plant health and quality and monitor physiological traits or diseases. However, detecting functional and degraded plant tissues in-vivo without harming the plant is extremely challenging. New solutions are needed in ligneous and perennial species, for which the sustainability of plantations is crucial. To tackle this challenge, we developed a novel approach based on multimodal 3D imaging and artificial intelligence-based image processing that allowed a non-destructive diagnosis of inner tissues in living plants. The method was successfully applied to the grapevine (Vitis vinifera L.). Vineyard's sustainability is threatened by trunk diseases, while the sanitary status of vines cannot be ascertained without injuring the plants. By combining MRI and X-ray CT 3D imaging with an automatic voxel classification, we could discriminate intact, degraded, and white rot tissues with a mean global accuracy of over 91%. Each imaging modality contribution to tissue detection was evaluated, and we identified quantitative structural and physiological markers characterizing wood degradation steps. The combined study of inner tissue distribution versus external foliar symptom history demonstrated that white rot and intact tissue contents are key-measurements in evaluating vines' sanitary status. We finally proposed a model for an accurate trunk disease diagnosis in grapevine. This work opens new routes for precision agriculture and in-situ monitoring of tissue quality and plant health across plant species., (© 2024. The Author(s).)

Effects of anthropogenic activities such as urbanization, population growth, and agriculture on water quality are major concerns particularly in low-income countries where water quality monitoring can be challenging. The purpose of the present study was to evaluate the cytogenotoxic potential of water from urban and rural Malagasy marshes, coupling a fish (Nile tilapia, Oreochromis niloticus) and a plant (Allium cepa) species as bioindicators. The fish and plants were exposed for 72 h to water sampled in the two locations investigated. Using the comet assay on fish erythrocytes, DNA strand breaks were assessed, while mitotic index and nucleolar alterations were estimated in cells of the plant root apex. Comet assays revealed significant DNA strand breaks to fish erythrocytes in both the marshes investigated while the mitotic index and nucleolar characteristics in the roots of A. cepa mainly highlighted potential cytotoxicity in the urban marsh. Our results demonstrate the advantages of coupling in vivo biological test systems to screen potential cytogenotoxicity of surface water in low-income countries where comprehensive data sets of aquatic contaminants are often lacking. Environ Toxicol Chem 2023;42:1266-1275. © 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC., (© 2023 The Authors. Environmental Toxicology and Chemistry published by Wiley Periodicals LLC on behalf of SETAC.)

Introduction: Differentiation of spore mother cells marks the somatic-to-reproductive transition in higher plants. Spore mother cells are critical for fitness because they differentiate into gametes, leading to fertilization and seed formation. The female spore mother cell is called the megaspore mother cell (MMC) and is specified in the ovule primordium. The number of MMCs varies by species and genetic background, but in most cases, only a single mature MMC enters meiosis to form the embryo sac. Multiple candidate MMC precursor cells have been identified in both rice and Arabidopsis , so variability in MMC number is likely due to conserved early morphogenetic events. In Arabidopsis , the restriction of a single MMC per ovule, or MMC singleness, is determined by ovule geometry. To look for potential conservation of MMC ontogeny and specification mechanisms, we undertook a morphogenetic description of ovule primordium growth at cellular resolution in the model crop maize., Methods: We generated a collection of 48 three-dimensional (3D) ovule primordium images for five developmental stages, annotated for 11 cell types. Quantitative analysis of ovule and cell morphological descriptors allowed the reconstruction of a plausible developmental trajectory of the MMC and its neighbors., Results: The MMC is specified within a niche of enlarged, homogenous L2 cells, forming a pool of candidate archesporial (MMC progenitor) cells. A prevalent periclinal division of the uppermost central archesporial cell formed the apical MMC and the underlying cell, a presumptive stack cell. The MMC stopped dividing and expanded, acquiring an anisotropic, trapezoidal shape. By contrast, periclinal divisions continued in L2 neighbor cells, resulting in a single central MMC., Discussion: We propose a model where anisotropic ovule growth in maize drives L2 divisions and MMC elongation, coupling ovule geometry with MMC fate., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ouedraogo, Lartaud, Baroux, Mosca, Delgado, Leblanc, Verdeil, Conéjéro and Autran.)

The growth and composition of fleshy fruits depend on resource acquisition and distribution in the plant. In tomato, the pedicel serves as the final connection between plant and fruit. However, very few quantitative data are available for the conducting tissues of the pedicel, nor is their genetic variability known. In the present study, a histological approach was combined with process-based modeling to evaluate the potential contribution made by the anatomy and histology of the pedicel to variations in fruit mass. Eleven genotypes were characterized and the impact of water deficit was studied for a single genotype using stress intensity and stage of application as variables. The results highlighted extensive variations in the relative proportions of the different pedicel tissues and in the absolute areas of xylem and phloem between genotypes. The model suggests that the variations in the area of the pedicel's vascular tissues induced by differences in genotype and water-deficit environments partly contributed to fruit mass variability. They therefore warrant phenotyping for use in the development of plant strains adapted to future environmental constraints. The results also demonstrated the need to develop non-invasive in vivo measurement methods to establish the number and size of active vessels and the flow rates in these vessels to improve prediction of water fluxes in plant architecture., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Background: Fruit abscission depends on cell separation that occurs within specialized cell layers that constitute an abscission zone (AZ). To determine the mechanisms of fleshy fruit abscission of the monocot oil palm (Elaeis guineensis Jacq.) compared with other abscission systems, we performed multi-scale comparative transcriptome analyses on fruit targeting the developing primary AZ and adjacent tissues., Results: Combining between-tissue developmental comparisons with exogenous ethylene treatments, and naturally occurring abscission in the field, RNAseq analysis revealed a robust core set of 168 genes with differentially regulated expression, spatially associated with the ripe fruit AZ, and temporally restricted to the abscission timing. The expression of a set of candidate genes was validated by qRT-PCR in the fruit AZ of a natural oil palm variant with blocked fruit abscission, which provides evidence for their functions during abscission. Our results substantiate the conservation of gene function between dicot dry fruit dehiscence and monocot fleshy fruit abscission. The study also revealed major metabolic transitions occur in the AZ during abscission, including key senescence marker genes and transcriptional regulators, in addition to genes involved in nutrient recycling and reallocation, alternative routes for energy supply and adaptation to oxidative stress., Conclusions: The study provides the first reference transcriptome of a monocot fleshy fruit abscission zone and provides insight into the mechanisms underlying abscission by identifying key genes with functional roles and processes, including metabolic transitions, cell wall modifications, signalling, stress adaptations and transcriptional regulation, that occur during ripe fruit abscission of the monocot oil palm. The transcriptome data comprises an original reference and resource useful towards understanding the evolutionary basis of this fundamental plant process.

Crown roots constitute the main part of the rice root system. Several key genes involved in crown root initiation and development have been identified by functional genomics approaches. Nevertheless, these approaches are impaired by functional redundancy and mutant lethality. To overcome these limitations, organ targeted transcriptome analysis can help to identify genes involved in crown root formation and early development. In this study, we generated an atlas of genes expressed in developing crown root primordia in comparison with adjacent stem cortical tissue at three different developmental stages before emergence, using laser capture microdissection. We identified 3975 genes differentially expressed in crown root primordia. About 30% of them were expressed at the three developmental stages, whereas 10.5%, 19.5% and 12.8% were specifically expressed at the early, intermediate and late stages, respectively. Sorting them by functional ontology highlighted an active transcriptional switch during the process of crown root primordia formation. Cross-analysis with other rice root development-related datasets revealed genes encoding transcription factors, chromatin remodeling factors, peptide growth factors, and cell wall remodeling enzymes that are likely to play a key role during crown root primordia formation. This atlas constitutes an open primary data resource for further studies on the regulation of crown root initiation and development., Competing Interests: J.L. is supported by a CIFRE fellowship (No: 2015/0195) from the Association Nationale de la Recherche Technologique, France, and a financial support from Biogemma, a subsidiary of the the seed company Limagrain. J.L., M.J., J.R. and C.S. are employees of the seed company Limagrain. This does not alter our adherence to PLOS ONE policies on sharing data and materials. There are no patents, products in development or marketed products associated with this research to declare.

Potassium (K + ) nutrition is of relevant interest for winegrowers because it influences grapevine growth, berry composition, as well as must and wine quality. Indeed, wine quality strongly depends on berry composition at harvest. However, K + content of grape berries increased steadily over the last decades, in part due to climate change. Currently, the properties and qualities of many fruits are also impacted by environment. In grapevine, this disturbs berry properties resulting in unbalanced wines with poor organoleptic quality and low acidity. This requires a better understanding of the molecular basis of K + accumulation and its control along grape berry development. This mini-review summarizes our current knowledge on K + nutrition in relation with fruit quality in the context of a changing environment., (Copyright © 2020 Villette, Cuéllar, Verdeil, Delrot and Gaillard.)

Grapevine (Vitis vinifera L.), one of the most important fruit crops, is a model plant for studying the physiology of fleshy fruits. Here, we report on the characterization of a new grapevine Shaker-type K+ channel, VvK5.1. Phylogenetic analysis revealed that VvK5.1 belongs to the SKOR-like subfamily. Our functional characterization of VvK5.1 in Xenopus oocytes confirms that it is an outwardly rectifying K+ channel that displays strict K+ selectivity. Gene expression level analyses by real-time quantitative PCR showed that VvK5.1 expression was detected in berries, roots, and flowers. In contrast to its Arabidopsis thaliana counterpart that is involved in K+ secretion in the root pericycle, allowing root to shoot K+ translocation, VvK5.1 expression territory is greatly enlarged. Using in situ hybridization we showed that VvK5.1 is expressed in the phloem and perivascular cells of berries and in flower pistil. In the root, in addition to being expressed in the root pericycle like AtSKOR, a strong expression of VvK5.1 is detected in small cells facing the xylem that are involved in lateral root formation. This fine and selective expression pattern of VvK5.1 at the early stage of lateral root primordia supports a role for outward channels to switch on cell division initiation., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Somatic embryogenesis (SE) is one of the most promising processes for large-scale dissemination of elite varieties. However, for many plant species, optimizing SE protocols still relies on a trial-and-error approach. Using coffee as a model plant, we report here the first global analysis of metabolome and hormone dynamics aiming to unravel mechanisms regulating cell fate and totipotency. Sampling from leaf explant dedifferentiation until embryo development covered 15 key stages. An in-depth statistical analysis performed on 104 metabolites revealed that massive re-configuration of metabolic pathways induced SE. During initial dedifferentiation, a sharp decrease in phenolic compounds and caffeine levels was also observed while auxins, cytokinins and ethylene levels were at their highest. Totipotency reached its highest expression during the callus stages when a shut-off in hormonal and metabolic pathways related to sugar and energetic substance hydrolysis was evidenced. Abscisic acid, leucine, maltotriose, myo-inositol, proline, tricarboxylic acid cycle metabolites and zeatin appeared as key metabolic markers of the embryogenic capacity. Combining metabolomics with multiphoton microscopy led to the identification of chlorogenic acids as markers of embryo redifferentiation. The present analysis shows that metabolite fingerprints are signatures of cell fate and represent a starting point for optimizing SE protocols in a rational way.

Fruitlet core rot is one of the major postharvest disease of pineapple ( Ananas comosus var. comosus ). In the past, control strategies were designed to eliminate symptoms without addressing their causes or mechanisms, thus achieving only moderate success. In this study, (i) we focused on the anatomy of the fruitlets in the resistant "MD-2" and susceptible "Queen" pineapple cultivars; (ii) we identified the key role of the carpel margin in the infection process; (iii) we identified the key role of the sinuous layer of thick-walled cells in the inhibition of Fusarium ananatum colonization; and (iv) we linked the anatomy of the fruitlets with the phenolic content of cell walls. The fruitlet anatomy of the two cultivars was studied using X-ray, fluorescence, and multiphoton microscopy. Sepals and bracts were not perfectly fused with each other, allowing the pathogen to penetrate the fruit even after flowering. In fact, the fungi were found in the blossom cups of both cultivars but only became pathogenic in the flesh of the "Queen" pineapple fruit under natural conditions. The outer layer of the "MD-2" cavity was continuous with thick cell walls composed of ferulic and coumaric acids. The cell walls of the "Queen" blossom cup were less lignified at the extremities, and the outer layer was interspersed with cracks. The carpel margins were fused broadly in the "MD-2" pineapple, in contrast to the "Queen" pineapple. This blemish allows the fungus to penetrate deeper into the susceptible cultivar. In pineapple fruitlets, the hyphae of F. ananatum mainly progressed directly between cell walls into the parenchyma but never reached the vascular region. A layer of thick-walled cells, in the case of the resistant cultivar, stopped the colonization, which were probably the infralocular septal nectaries. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to F. ananatum . The major phenolics bound to the cell walls were coumaric and ferulic acids and were found in higher amounts in the resistant cultivar postinoculation. The combination of fruitlet anatomy and lignification plays a role in the mechanism of host resistance to fruitlet core rot.

Today, in the presence of global warming, understanding how plants respond to drought stress is essential to meet the challenge of developing new cultivars and new irrigation strategies, consistent with the maintenance of crop productivity. In this context, the study of the relation between plants and water is of central interest for modeling their responses to biotic and abiotic constraints. Paradoxically, there are very few direct and noninvasive methods to quantify and measure the level and the flow of water in plants. The present work aims to develop a noninvasive methodology for living plant based on nuclear magnetic resonance (NMR) at low magnetic field and imaging (MRI) to tackle the issue of water quantity in plants. For this purpose, a portable NMR device measuring the signal level at 8 mT was built. This instrument addresses specific challenges such as miniaturization, accessibility, and overheating in order to maintain the plant intact of time over long period. Time dependence of the water content in sorghum plants is reported under abiotic stress as well as the fraction of transpirable soil water and the photosynthesis activity through the leaves. At high magnetic field (9.4 T), T2 maps were acquired on the same sorghum plants at two time points. The combination of these approaches allows us to identify ecophysiological biomarkers of drought stress. One particular interesting result concerns the spatial distribution of water in two anatomically contrasted sorghum genotypes., (© 2019 John Wiley & Sons, Ltd.)

In grapevine, climate changes lead to increased berry potassium (K + ) contents that result in must with low acidity. Consequently, wines are becoming 'flat' to the taste, with poor organoleptic properties and low potential aging, resulting in significant economic loss. Precise investigation into the molecular determinants controlling berry K + accumulation during its development are only now emerging. Here, we report functional characterization by electrophysiology of a new grapevine Shaker-type K + channel, VvK3.1. The analysis of VvK3.1 expression patterns was performed by qPCR and in situ hybridization. We found that VvK3.1 belongs to the AKT2 channel phylogenetic branch and is a weakly rectifying channel, mediating both inward and outward K + currents. We showed that VvK3.1 is highly expressed in the phloem and in a unique structure located at the two ends of the petiole, identified as a pulvinus. From the onset of fruit ripening, all data support the role of the VvK3.1 channel in the massive K + fluxes from the phloem cell cytosol to the berry apoplast during berry K + loading. Moreover, the high amount of VvK3.1 transcripts detected in the pulvinus strongly suggests a role for this Shaker in the swelling and shrinking of motor cells involved in paraheliotropic leaf movements., (© 2018 The Authors. New Phytologist © 2018 New Phytologist Trust.)

The climate warming implies an increase of stress of plants (drought and torrential rainfall). The understanding of plant behavior, in this context, takes a major importance and sap flow measurement in plants remains a key issue for plant understanding. Magnetic Resonance Imaging (MRI) which is well known to be a powerful tool to access water quantity can be used to measure moving water. We describe a novel flow-MRI method which takes advantage of inflow slice sensitivity. The method involves the slice selectivity in the context of multi slice spin echo sequence. Two sequences such as a given slice is consecutively inflow and outflow sensitive are performed, offering the possiblility to perform slow flow sensitive imaging in a quite straigthforward way. The method potential is demonstrated by imaging both a slow flow measurement on a test bench (as low as 10 μm.s-1) and the Poiseuille's profile of xylemian sap flow velocity in the xylematic tissues of a tomato plant stem.

Cocoa self-compatibility is an important yield factor and has been described as being controlled by a late gameto-sporophytic system expressed only at the level of the embryo sac. It results in gametic non-fusion and involves several loci. In this work, we identified two loci, located on chromosomes 1 and 4 (CH1 and CH4), involved in cocoa self-incompatibility by two different processes. Both loci are responsible for gametic selection, but only one (the CH4 locus) is involved in the main fruit drop. The CH1 locus acts prior to the gamete fusion step and independently of the CH4 locus. Using fine-mapping and genome-wide association studies, we focused analyses on restricted regions and identified candidate genes. Some of them showed a differential expression between incompatible and compatible reactions. Immunolocalization experiments provided evidence of CH1 candidate genes expressed in ovule and style tissues. Highly polymorphic simple sequence repeat (SSR) diagnostic markers were designed in the CH4 region that had been identified by fine-mapping. They are characterized by a strong linkage disequilibrium with incompatibility alleles, thus allowing the development of efficient diagnostic markers predicting self-compatibility and fruit setting according to the presence of specific alleles or genotypes. SSR alleles specific to self-compatible Amelonado and Criollo varieties were also identified, thus allowing screening for self-compatible plants in cocoa populations., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The oil palm ( Elaeis guineensis ), a monocotyledonous species in the family Arecaceae, has an extraordinarily oil rich fleshy mesocarp, and presents an original model to examine the ripening processes and regulation in this particular monocot fruit. Histochemical analysis and cell parameter measurements revealed cell wall and middle lamella expansion and degradation during ripening and in response to ethylene. Cell wall related transcript profiles suggest a transition from synthesis to degradation is under transcriptional control during ripening, in particular a switch from cellulose, hemicellulose, and pectin synthesis to hydrolysis and degradation. The data provide evidence for the transcriptional activation of expansin, polygalacturonase, mannosidase, beta-galactosidase, and xyloglucan endotransglucosylase/hydrolase proteins in the ripening oil palm mesocarp, suggesting widespread conservation of these activities during ripening for monocotyledonous and eudicotyledonous fruit types. Profiling of the most abundant oil palm polygalacturonase ( EgPG4 ) and 1-aminocyclopropane-1-carboxylic acid oxidase ( ACO ) transcripts during development and in response to ethylene demonstrated both are sensitive markers of ethylene production and inducible gene expression during mesocarp ripening, and provide evidence for a conserved regulatory module between ethylene and cell wall pectin degradation. A comprehensive analysis of NAC transcription factors confirmed at least 10 transcripts from diverse NAC domain clades are expressed in the mesocarp during ripening, four of which are induced by ethylene treatment, with the two most inducible ( EgNAC6 and EgNAC7 ) phylogenetically similar to the tomato NAC-NOR master-ripening regulator. Overall, the results provide evidence that despite the phylogenetic distance of the oil palm within the family Arecaceae from the most extensively studied monocot banana fruit, it appears ripening of divergent monocot and eudicot fruit lineages are regulated by evolutionarily conserved molecular physiological processes.

We report here the isolation and functional analysis of AlTMP1 gene encoding a member of the PMP3 protein family. In Aeluropus littoralis , AlTMP1 is highly induced by abscisic acid (ABA), cold, salt, and osmotic stresses. Transgenic tobacco expressing AlTMP1 exhibited enhanced tolerance to salt, osmotic, H₂O₂, heat and freezing stresses at the seedling stage. Under greenhouse conditions, the transgenic plants showed a higher level of tolerance to drought than to salinity. Noteworthy, AlTMP1 plants yielded two- and five-fold more seeds than non-transgenic plants (NT) under salt and drought stresses, respectively. The leaves of AlTMP1 plants accumulated lower Na⁺ but higher K⁺ and Ca 2+ than those of NT plants. Tolerance to osmotic and salt stresses was associated with higher membrane stability, low electrolyte leakage, and improved water status. Finally, accumulation of AlTMP1 in tobacco altered the regulation of some stress-related genes in either a positive ( NHX1 , CAT1 , APX1 , and DREB1A ) or negative ( HKT1 and KT1 ) manner that could be related to the observed tolerance. These results suggest that AlTMP1 confers stress tolerance in tobacco through maintenance of ion homeostasis, increased membrane integrity, and water status. The observed tolerance may be due to a direct or indirect effect of AlTMP1 on the expression of stress-related genes which could stimulate an adaptive potential not present in NT plants.

Pearl millet plays an important role for food security in arid regions of Africa and India. Nevertheless, it is considered an orphan crop as it lags far behind other cereals in terms of genetic improvement efforts. Breeding pearl millet varieties with improved root traits promises to deliver benefits in water and nutrient acquisition. Here, we characterize early pearl millet root system development using several different root phenotyping approaches that include rhizotrons and microCT. We report that early stage pearl millet root system development is characterized by a fast growing primary root that quickly colonizes deeper soil horizons. We also describe root anatomical studies that revealed three distinct types of lateral roots that form on both primary roots and crown roots. Finally, we detected significant variation for two root architectural traits, primary root lenght and lateral root density, in pearl millet inbred lines. This study provides the basis for subsequent genetic experiments to identify loci associated with interesting early root development traits in this important cereal.

The oil palm (Elaeis guineensis Jacq.) fruit primary abscission zone (AZ) is a multi-cell layered boundary region between the pedicel (P) and mesocarp (M) tissues. To examine the cellular processes that occur during the development and function of the AZ cell layers, we employed multiple histological and immunohistochemical methods combined with confocal, electron and Fourier-transform infrared (FT-IR) microspectroscopy approaches. During early fruit development and differentiation of the AZ, the orientation of cell divisions in the AZ was periclinal compared with anticlinal divisions in the P and M. AZ cell wall width increased earlier during development suggesting cell wall assembly occurred more rapidly in the AZ than the adjacent P and M tissues. The developing fruit AZ contain numerous intra-AZ cell layer plasmodesmata (PD), but very few inter-AZ cell layer PD. In the AZ of ripening fruit, PD were less frequent, wider, and mainly intra-AZ cell layer localized. Furthermore, DAPI staining revealed nuclei are located adjacent to PD and are remarkably aligned within AZ layer cells, and remain aligned and intact after cell separation. The polarized accumulation of ribosomes, rough endoplasmic reticulum, mitochondria, and vesicles suggested active secretion at the tip of AZ cells occurred during development which may contribute to the striated cell wall patterns in the AZ cell layers. AZ cells accumulated intracellular pectin during development, which appear to be released and/or degraded during cell separation. The signal for the JIM5 epitope, that recognizes low methylesterified and un-methylesterified homogalacturonan (HG), increased in the AZ layer cell walls prior to separation and dramatically increased on the separated AZ cell surfaces. Finally, FT-IR microspectroscopy analysis indicated a decrease in methylesterified HG occurred in AZ cell walls during separation, which may partially explain an increase in the JIM5 epitope signal. The results obtained through a multi-imaging approach allow an integrated view of the dynamic developmental processes that occur in a multi-layered boundary AZ and provide evidence for distinct regulatory mechanisms that underlie oil palm fruit AZ development and function.

Background: Drought is a widespread limiting factor in coffee plants. It affects plant development, fruit production, bean development and consequently beverage quality. Genetic diversity for drought tolerance exists within the coffee genus. However, the molecular mechanisms underlying the adaptation of coffee plants to drought are largely unknown. In this study, we compared the molecular responses to drought in two commercial cultivars (IAPAR59, drought-tolerant and Rubi, drought-susceptible) of Coffea arabica grown in the field under control (irrigation) and drought conditions using the pyrosequencing of RNA extracted from shoot apices and analysing the expression of 38 candidate genes., Results: Pyrosequencing from shoot apices generated a total of 34.7 Mbp and 535,544 reads enabling the identification of 43,087 clusters (41,512 contigs and 1,575 singletons). These data included 17,719 clusters (16,238 contigs and 1,575 singletons) exclusively from 454 sequencing reads, along with 25,368 hybrid clusters assembled with 454 sequences. The comparison of DNA libraries identified new candidate genes (n = 20) presenting differential expression between IAPAR59 and Rubi and/or drought conditions. Their expression was monitored in plagiotropic buds, together with those of other (n = 18) candidates genes. Under drought conditions, up-regulated expression was observed in IAPAR59 but not in Rubi for CaSTK1 (protein kinase), CaSAMT1 (SAM-dependent methyltransferase), CaSLP1 (plant development) and CaMAS1 (ABA biosynthesis). Interestingly, the expression of lipid-transfer protein (nsLTP) genes was also highly up-regulated under drought conditions in IAPAR59. This may have been related to the thicker cuticle observed on the abaxial leaf surface in IAPAR59 compared to Rubi., Conclusions: The full transcriptome assembly of C. arabica, followed by functional annotation, enabled us to identify differentially expressed genes related to drought conditions. Using these data, candidate genes were selected and their differential expression profiles were confirmed by qPCR experiments in plagiotropic buds of IAPAR59 and Rubi under drought conditions. As regards the genes up-regulated under drought conditions, specifically in the drought-tolerant IAPAR59, several corresponded to orphan genes but also to genes coding proteins involved in signal transduction pathways, as well as ABA and lipid metabolism, for example. The identification of these genes should help advance our understanding of the genetic determinism of drought tolerance in coffee.

Root and stem rot (RSR) disease caused by Fusarium oxysporum f. sp. radicis-vanillae (Forv) is the most damaging disease of vanilla (Vanilla planifolia and V. × tahitensis, Orchidaceae). Breeding programs aimed at developing resistant vanilla varieties are hampered by the scarcity of sources of resistance to RSR and insufficient knowledge about the histopathology of Forv. In this work we have (i) identified new genetic resources resistant to RSR including V. planifolia inbreds and vanilla relatives, (ii) thoroughly described the colonization pattern of Forv into selected vanilla accessions, confirming its necrotic non-vascular behavior in roots, and (iii) evidenced the key role played by hypodermis, and particularly lignin deposition onto hypodermal cell walls, for resistance to Forv in two highly resistant vanilla accessions. Two hundred and fifty-four vanilla accessions were evaluated in the field under natural conditions of infection and in controlled conditions using in vitro plants root-dip inoculated by the highly pathogenic isolate Fo072. For the 26 accessions evaluated in both conditions, a high correlation was observed between field evaluation and in vitro assay. The root infection process and plant response of one susceptible and two resistant accessions challenged with Fo072 were studied using wide field and multiphoton microscopy. In susceptible V. planifolia, hyphae penetrated directly into the rhizodermis in the hairy root region then invaded the cortex through the passage cells where it induced plasmolysis, but never reached the vascular region. In the case of the resistant accessions, the penetration was stopped at the hypodermal layer. Anatomical and histochemical observations coupled with spectral analysis of the hypodermis suggested the role of lignin deposition in the resistance to Forv. The thickness of lignin constitutively deposited onto outer cell walls of hypodermis was highly correlated with the level of resistance for 21 accessions tested. The accumulation of p-coumaric and sinapic acids, two phenolic precursors of lignin, was observed in the resistant plants inoculated with Fo072, but not in the susceptible one. Altogether, our analyses enlightened the mechanisms at work in RSR resistant genotypes and should enhance the development of novel breeding strategies aimed at improving the genetic control of RSR of vanilla.

Key Message: When fused to " Pr AlSAP " promoter, transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Pr AlSAP can be used for engineering abiotic stress tolerance. We previously showed that ectopic expression of a stress-associated protein gene from Aeluropus littoralis (AlSAP) enhances tolerance to multiple abiotic stresses in tobacco, wheat and rice. The ortholog of AlSAP in rice is OsSAP9. Here, we demonstrate that AlSAP transcripts accumulate in Aeleuropus in response to multiple abiotic stresses and at a higher level in roots, while those of OsSAP9 are preferentially induced by cold and heat treatments and accumulate preferentially in leaves of rice. In silico analysis of the AlSAP promoter "Pr AlSAP " predicted several cis-acting elements responsible for gene regulation by dehydration, salt, heat, ABA, SA, wounding and tissue-specific expression. The Pr AlSAP promoter was fused to the gusA gene and used to produce transgenic rice plants. Transcripts of gusA exhibited similar accumulation patterns in transgenic rice as AlSAP transcripts in A. littoralis. Indeed, accumulation of gusA transcripts was higher in roots than in leaves and induced by salt, drought, cold and heat treatments. GUS activity was confirmed in roots, coleoptiles, leaves and glumes, but absent in the root cell elongation zone and in dry seeds. A wound treatment strongly induced GUS accumulation in leaves and imbibed seeds. Altogether, these results indicate that the regulatory regions of two ortholog genes "AlSAP" and "OsSAP9" have diverged in the specificity of the signals promoting their induction, but that the trans-acting elements allowing the correct spatiotemporal regulation and stress induction of Pr AlSAP exist in rice. Therefore, the AlSAP promoter appears to be an interesting candidate for engineering abiotic stress tolerance in cereals.

Autofluorescent molecules are abundant in plant cells and spectral images offer means for analyzing their spectra, yielding information on their accumulation and function. Based on their fluorescence characteristics, an imaging approach using multiphoton microscopy was designed to assess localization of the endogenous fluorophores in living plant cells. This method, which requires no previous treatment, provides an effective experimental tool for discriminating between multiple naturally-occurring fluorophores in living-tissues. Combined with advanced Linear Unmixing, the spectral analysis extends the possibilities and enables the simultaneous detection of fluorescent molecules reliably separating overlapping emission spectra. However, as with any technology, the possibility for artifactual results does exist. This methodological article presents an overview of the applications of tissular and intra-cellular localization of these intrinsic fluorophores in leaves and fruits (here for coffee and vanilla). This method will provide new opportunities for studying cellular environments and the behavior of endogenous fluorophores in the intracellular environment.

We developed the PHIV-RootCell software to quantify anatomical traits of rice roots transverse section images. Combined with an efficient root sample processing method for image acquisition, this program permits supervised measurements of areas (those of whole root section, stele, cortex, and central metaxylem vessels), number of cell layers and number of cells per cell layer. The PHIV-RootCell toolset runs under ImageJ, an independent operating system that has a license-free status. To demonstrate the usefulness of PHIV-RootCell, we conducted a genetic diversity study and an analysis of salt stress responses of root anatomical parameters in rice (Oryza sativa L.). Using 16 cultivars, we showed that we could discriminate between some of the varieties even at the 6 day-olds stage, and that tropical japonica varieties had larger root sections due to an increase in cell number. We observed, as described previously, that root sections become enlarged under salt stress. However, our results show an increase in cell number in ground tissues (endodermis and cortex) but a decrease in external (peripheral) tissues (sclerenchyma, exodermis, and epidermis). Thus, the PHIV-RootCell program is a user-friendly tool that will be helpful for future genetic and physiological studies that investigate root anatomical trait variations.

A multiple cell imaging approach combining immunofluorescence by confocal microscopy, fluorescence spectral analysis by multiphotonic microscopy, and transmission electron microscopy identified the site of accumulation of 4-O-(3-methoxybenzaldehyde) β-d-glucoside, a phenol glucoside massively stockpiled by vanilla fruit. The glucoside is sufficiently abundant to be detected by spectral analysis of its autofluorescence. The convergent results obtained by these different techniques demonstrated that the phenol glucoside accumulates in the inner volume of redifferentiating chloroplasts as solid amorphous deposits, thus ensuring phenylglucoside cell homeostasis. Redifferentiation starts with the generation of loculi between thylakoid membranes which are progressively filled with the glucoside until a fully matured organelle is obtained. This peculiar mode of storage of a phenolic secondary metabolite is suspected to occur in other plants and its generalization in the Plantae could be considered. This new chloroplast-derived organelle is referred to as a 'phenyloplast'., (© The Author 2014. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

In a previous work, we demonstrated that expression of TdPIP2;1 in Xenopus oocytes resulted in an increase in Pf compared to water injected oocytes. Phenotypic analyses of transgenic tobacco plants expressing TdPIP2;1 generated a tolerance phenotype towards drought and salinity stresses. To elucidate its stress tolerance mechanism at the transcriptional level, we isolated and characterized the promoter region of the TdPIP2;1 gene. A 1060-bp genomic fragment upstream of the TdPIP2;1 translated sequence has been isolated, cloned, and designated as the proTdPIP2;1 promoter. Sequence analysis of proTdPIP2;1 revealed the presence of cis regulatory elements which could be required for abiotic stress responsiveness, for tissue-specific and vascular expression. The proTdPIP2;1 promoter was fused to the β-glucuronidase (gusA) gene and the resulting construct was transferred into rice (cv. Nipponbare). Histochemical analysis of proTdPIP2;1::Gus in rice plants revealed that the GUS activity was observed in leaves, stems and roots of stably transformed rice T3 plants. Histological sections prepared revealed accumulation of GUS products in phloem, xylem and in some cells adjacent to xylem. The transcripts were up-regulated by dehydration. Transgenic rice plants overexpressing proTdPIP2;1 in fusion with TdPIP2;1, showed enhanced drought tolerance, while wild type plants were more sensitive and exhibited symptoms of wilting and chlorosis. These findings suggest that expression of the TdPIP2;1 gene regulated by its own promoter achieves enhanced drought tolerance in rice., (Copyright © 2014 Elsevier Masson SAS. All rights reserved.)

In this work, we studied the impact of the successive steps of the droplet-vitrification protocol technique employed for cryopreservation of Rubia akane hairy roots on the features of cortical, pericycle and endoderm cells of apical and central root segments, using histology techniques and combining qualitative and quantitative observations. In apical segments, plasmolysis (22-71 %, depending on cell type) was observed only after the loading treatment and did not increase after the following steps of the protocol. By contrast, in central segments, plasmolysis (39-45 %) was already observed after the sucrose pretreatment; it increased to 54-68 %, depending on cell type, after the loading treatment, but no further changes were noted after treatment with the vitrification solution. After liquid nitrogen exposure and unloading treatment, deplasmolysis was more rapid in apical segments, with cortical and pericycle cells having retrieved their original features. In central segments, only cortical cells had retrieved their original features and endoderm and pericycle cells were still highly plasmolysed. Nuclei were more strongly impacted by the cryopreservation protocol in central segments, where they displayed a highly condensed nucleoplasm from the loading treatment onwards and had not retrieved their original aspect after the unloading treatment. By contrast, nuclei had a much less condensed nucleoplasm in cells of apical segments, and they had retrieved their original aspect after the unloading treatment.

An imaging method using spectral analysis combined with advanced linear unmixing was used to allow histolocalization of natural autofluorescent compounds such as hydroxycinnamic acid (chlorogenic acid) and xanthone (mangiferin) in living cells and tissues (mature coffee leaves). The tested method included three complementary steps: 1/ visualization of natural autofluorescence and spectrum acquisition with a multiphoton microscope; 2/ identification of some compounds using previous information on the chemical composition of the tissue, obtained from litterature; and 3/ localization of candidate compounds by spectral imaging. The second part of the study consisted of describing the histochemical structure of leaves during their development. This revealed very fast histochemical differentiation of leaves during the first week after their emergence. Lastly, young leaves of Coffea pseudozanguebariae (PSE), C. eugenioides (EUG), C. arabica (ARA) and C. canephora (CAN) were compared. This confirmed the presence of xanthone in PSE and EUG, but especially its precise tissue localization. This also highlighted the paternal CAN origin of the leaf structure in the allotetraploid species ARA. The limits and advantages of the method without staining are discussed relative to classical epifluorescence microscopy under UV light. This non-invasive optical technique does not require pretreatment and is an effective experimental tool to differentiate multiple naturally-occuring fluorochores in living tissues.

In this work, we performed qualitative and quantitative observations of the cytological changes occurring in cells of yam (Dioscorea alata) in vitro shoot tips cryopreserved using the encapsulation-dehydration (E-D) technique. Shoot tip osmoprotection for 24 h in 1.25 M sucrose medium induced drastic changes in cellular cytological features, including high plasmolysis in all three cellular areas studied, the external cell layer (L1), one to three (L1-3) and seven to nine (L7-9) cell layers from the surface of the meristematic dome, pyknotic nuclei in meristematic area cells and disappearance of nucleoli. Nucleus size decreased significantly in all cellular areas studied. Nucleocytoplasmic ratio decreased significantly in L1-3 and L7-9 cells. Nuclear protein content increased, particularly in L1 and L1-3 cells. After physical dehydration, plasma membrane of numerous basal part cells was broken and intracellular soluble protein leakage was observed. Nucleus area and nucleocytoplasmic ratio decreased significantly in L7-9 cells. One week after cryopreservation, shoot tips showed regrowth and living cells had recovered their original morphology. In all cellular areas studied, nuclei had retrieved their original staining and nucleoli were visible. Original nucleus area values were recovered in L1-3 and L1 cells. The nucleocytoplasmic ratio retrieved its initial value in L1 cells but remained at levels observed after osmoprotection for L1-3 and L7-9 cells. The nuclear protein content had retrieved its original level. This investigation provided new insights in changes occurring in D. alata apices throughout an E-D protocol.

Background and Aims: Condensed tannins (also called proanthocyanidins) are widespread polymers of catechins and are essential for the defence mechanisms of vascular plants (Tracheophyta). A large body of evidence argues for the synthesis of monomeric epicatechin on the cytosolic face of the endoplasmic reticulum and its transport to the vacuole, although the site of its polymerization into tannins remains to be elucidated. The aim of the study was to re-examine the cellular frame of tannin polymerization in various representatives of the Tracheophyta., Methods: Light microscopy epifluorescence, confocal microscopy, transmission electron microscopy (TEM), chemical analysis of tannins following cell fractionation, and immunocytochemistry were used as independent methods on tannin-rich samples from various organs from Cycadophyta, Ginkgophyta, Equisetophyta, Pteridophyta, Coniferophyta and Magnoliophyta. Tissues were fixed in a caffeine-glutaraldehyde mixture and examined by TEM. Other fresh samples were incubated with primary antibodies against proteins from both chloroplastic envelopes and a thylakoidal chlorophyll-carrying protein; they were also incubated with gelatin-Oregon Green, a fluorescent marker of condensed tannins. Coupled spectral analyses of chlorophyll and tannins were carried out by confocal microscopy on fresh tissues and tannin-rich accretions obtained through cell fractionation; chemical analyses of tannins and chlorophylls were also performed on the accretions., Key Results and Conclusions: The presence of the three different chloroplast membranes inside vacuolar accretions that constitute the typical form of tannin storage in vascular plants was established in fresh tissues as well as in purified organelles, using several independent methods. Tannins are polymerized in a new chloroplast-derived organelle, the tannosome. These are formed by pearling of the thylakoids into 30 nm spheres, which are then encapsulated in a tannosome shuttle formed by budding from the chloroplast and bound by a membrane resulting from the fusion of both chloroplast envelopes. The shuttle conveys numerous tannosomes through the cytoplasm towards the vacuole in which it is then incorporated by invagination of the tonoplast. Finally, shuttles bound by a portion of tonoplast aggregate into tannin accretions which are stored in the vacuole. Polymerization of tannins occurs inside the tannosome regardless of the compartment being crossed. A complete sequence of events apparently valid in all studied Tracheophyta is described.

Deciphering cellular iron (Fe) homeostasis requires having access to both quantitative and qualitative information on the subcellular pools of Fe in tissues and their dynamics within the cells. We have taken advantage of the Perls/DAB Fe staining procedure to perform a systematic analysis of Fe distribution in roots, leaves and reproductive organs of the model plant Arabidopsis thaliana, using wild-type and mutant genotypes affected in iron transport and storage. Roots of soil-grown plants accumulate iron in the apoplast of the central cylinder, a pattern that is strongly intensified when the citrate effluxer FRD3 is not functional, thus stressing the importance of citrate in the apoplastic movement of Fe. In leaves, Fe level is low and only detected in and around vascular tissues. In contrast, Fe staining in leaves of iron-treated plants extends in the surrounding mesophyll cells where Fe deposits, likely corresponding to Fe-ferritin complexes, accumulate in the chloroplasts. The loss of ferritins in the fer1,3,4 triple mutant provoked a massive accumulation of Fe in the apoplastic space, suggesting that in the absence of iron buffering in the chloroplast, cells activate iron efflux and/or repress iron influx to limit the amount of iron in the cell. In flowers, Perls/DAB staining has revealed a major sink for Fe in the anthers. In particular, developing pollen grains accumulate detectable amounts of Fe in small-size intracellular bodies that aggregate around the vegetative nucleus at the binuclear stage and that were identified as amyloplasts. In conclusion, using the Perls/DAB procedure combined to selected mutant genotypes, this study has established a reliable atlas of Fe distribution in the main Arabidopsis organs, proving and refining long-assumed intracellular locations and uncovering new ones. This "iron map" of Arabidopsis will serve as a basis for future studies of possible actors of iron movement in plant tissues and cell compartments.

In Coffea arabica L., the development of direct sowing of somatic embryos (SE) in planting substrate, with subsequent nursery production of plants, has promoted the industrialization of somatic embryogenesis. However, plant conversion rates are still low and require improvements to enhance the cost-effectiveness of commercial micropropagation. With the aim of improving plant regeneration from SE, we studied the morphological and histological criteria and water characteristics during germination and plant conversion of zygotic embryos (ZE) and SE. At the cotyledonary stage, SE produced in a 1 l RITA(®) temporary immersion bioreactor (area 55.8 cm(2)) were morphologically similar in size (2-3 mm) but abnormal as compared with mature ZE. Protein and starch reserve levels were extremely low throughout germination and conversion to plantlets, while the water status remained steady [water content (WC) from 76 to 87%, Ψ from -0.37 to -0.47 MPa, pressure potential from 0.69 to 0.24 MPa]. In ZE, spectacular hydration occurred during the first 3 weeks (WC from 37 to 75%; Ψ from -6.24 to -1.0 MPa). Cotyledons remained undifferentiated for 10 weeks after sowing. Conversely, after only 3 weeks under germination conditions in a RITA(®) bioreactor, spongy and palisade parenchyma and stomata formed in SE cotyledons. The ZE plant conversion was faster than that of SE (14 vs. 22 weeks) and more efficient (rates 96 vs. 55%), with much more substantial hypocotyl and cotyledon development. The use of a new 5 l MATIS(®) bioreactor (area 355 cm(2)), designed especially to favor embryo dispersion and light transmittance to SE, markedly improved the embryo-to-plantlet conversion rate (91%). These results highlight the morphological heterogeneity and lack of protein reserves in SE at the beginning of the germination phase and marked differences in water characteristics. However, they also reveal high phenotypic plasticity, leading to a highly efficient plantlet conversion rate due to better embryo dispersion and light transmittance in more horizontal bioreactors.

The grape berry provides a model for investigating the physiology of non-climacteric fruits. Increased K(+) accumulation in the berry has a strong negative impact on fruit acidity (and quality). In maturing berries, we identified a K(+) channel from the Shaker family, VvK1.2, and two CBL-interacting protein kinase (CIPK)/calcineurin B-like calcium sensor (CBL) pairs, VvCIPK04-VvCBL01 and VvCIPK03-VvCBL02, that may control the activity of this channel. VvCBL01 and VvCIPK04 are homologues of Arabidopsis AtCBL1 and AtCIPK23, respectively, which form a complex that controls the activity of the Shaker K(+) channel AKT1 in Arabidopsis roots. VvK1.2 remained electrically silent when expressed alone in Xenopus oocytes, but gave rise to K(+) currents when co-expressed with the pairs VvCIPK03-VvCBL02 or VvCIPK04-VvCBL01, the second pair inducing much larger currents than the first one. Other tested CIPK-CBL pairs expressed in maturing berries were found to be unable to activate VvK1.2. When activated by its CIPK-CBL partners, VvK1.2 acts as a voltage-gated inwardly rectifying K(+) channel that is activated at voltages more negative than -100 mV and is stimulated upon external acidification. This channel is specifically expressed in the berry, where it displays a very strong induction at veraison (the inception of ripening) in flesh cells, phloem tissues and perivascular cells surrounding vascular bundles. Its expression in these tissues is further greatly increased upon mild drought stress. VvK1.2 is thus likely to mediate rapid K(+) transport in the berry and to contribute to the extensive re-organization of the translocation pathways and transport mechanisms that occurs at veraison., (© 2012 The Authors The Plant Journal © 2012 Blackwell Publishing Ltd.)

Background: Cell separation that occurs during fleshy fruit abscission and dry fruit dehiscence facilitates seed dispersal, the final stage of plant reproductive development. While our understanding of the evolutionary context of cell separation is limited mainly to the eudicot model systems tomato and Arabidopsis, less is known about the mechanisms underlying fruit abscission in crop species, monocots in particular. The polygalacturonase (PG) multigene family encodes enzymes involved in the depolymerisation of pectin homogalacturonan within the primary cell wall and middle lamella. PG activity is commonly found in the separation layers during organ abscission and dehiscence, however, little is known about how this gene family has diverged since the separation of monocot and eudicots and the consequence of this divergence on the abscission process., Results: The objective of the current study was to identify PGs responsible for the high activity previously observed in the abscission zone (AZ) during fruit shedding of the tropical monocot oil palm, and to analyze PG gene expression during oil palm fruit ripening and abscission. We identified 14 transcripts that encode PGs, all of which are expressed in the base of the oil palm fruit. The accumulation of five PG transcripts increase, four decrease and five do not change during ethylene treatments that induce cell separation. One PG transcript (EgPG4) is the most highly induced in the fruit base, with a 700-5000 fold increase during the ethylene treatment. In situ hybridization experiments indicate that the EgPG4 transcript increases preferentially in the AZ cell layers in the base of the fruit in response to ethylene prior to cell separation., Conclusions: The expression pattern of EgPG4 is consistent with the temporal and spatial requirements for cell separation to occur during oil palm fruit shedding. The sequence diversity of PGs and the complexity of their expression in the oil palm fruit tissues contrast with data from tomato, suggesting functional divergence underlying the ripening and abscission processes has occurred between these two fruit species. Furthermore, phylogenetic analysis of EgPG4 with PGs from other species suggests some conservation, but also diversification has occurred between monocots and eudicots, in particular between dry and fleshy fruit species.

Background and Aims: Oil palm, an unbranched perennial monocotyledon, possesses a single shoot apical meristem (SAM), which is responsible for the initiation of the entire above-ground structure of the plant. To compare the palm SAM structure with those of other monocots and to study variations in its structure throughout the life of the plant, its organization was characterized from the embryonic stage to that of the reproductive plant., Methods: SAM structure was studied by a combination of stained histological sections, light and confocal microscopy, and serial section-based three-dimensional reconstructions., Key Results: The oil palm SAM is characterized by two developmental phases: a juvenile phase with a single tunica-corpus structure displaying a gradual increase in size; and a mature phase characterized by a stable size, a modified shape and an established histological zonation pattern. In mature plants, fluctuations in SAM shape and volume occur, mainly as a consequence of changes in the central zone, possibly in relation to leaf initiation., Conclusions: Development of the oil palm SAM is characterized by a juvenile to mature phase transition accompanied by establishment of a zonal pattern and modified shape. SAM zonation is dynamic during the plastochron period and displays distinct features compared with other monocots.

The biochemical composition of leaves from Coffea pseudozanguebariae, a wild caffeine-free coffee species, was determined. Two phenolic compounds were extracted from leaves, separated and characterized. Their structures were elucidated by mass spectrometry, and 1D and 2D NMR spectroscopy and were shown to be mangiferin (1) and isomangiferin (2), which were the main polyphenol products. Multiphoton fluorescence imaging was performed to visualize polyphenol distribution in leaf cross sections. Consistent biochemical analysis cell imaging techniques on leaves revealed yellow fluorescence in the epidermis and parenchyma cells corresponding to xanthone compounds.

Global DNA methylation was assessed by high-performance liquid chromatography (HPLC) for the first time in Eucalyptus urophylla×Eucalyptus grandis shoot tips comparing three outdoor and one in vitro sources of related genotypes differing in their physiological age. The DNA methylation levels found were consistent with those reported for other Angiosperms using the same HPLC technology. Notwithstanding noticeable time-related fluctuations within each source of plant material, methylation rate was overall higher for the mature clone (13.7%) than for the rejuvenated line of the same clone (12.6%) and for the juvenile offspring seedlings (11.8%). The in vitro microshoots of the mature clone were less methylated (11.3%) than the other outdoor origins, but the difference with the juvenile seedlings was not significant. Immunofluorescence investigations on shoot apices established that the mature source could be distinguished from the rejuvenated and juvenile origins by a higher density of cells with methylated nuclei in leaf primordia. Shoot apical meristems (SAMs) from the mature clone also showed a greater proportion and more methylated cells than SAMs from the rejuvenated and juvenile origins. The nuclei of these latter were characterized by fewer and more dispersed labeled spots than for the mature source. Our findings establish that physiological ageing induced quantitative and qualitative variations of DNA methylation at shoot tip, SAM and even cellular levels. Overall this DNA methylation increased with maturation and conversely decreased with rejuvenation to reach the lower scores and to show the immunolabeling patterns that characterized juvenile material nuclei., (Copyright © Physiologia Plantarum 2011.)

In our recent published work it has been demonstrated that AlSAP, a gene encoding an A20/AN1 zinc-finger protein (stress-associated protein) of the C4 halophyte grass Aeluropus littoralis, is inducible by various abiotic stresses and by hormonal stimuli. To further investigate the regulation of the gene, a 586-bp genomic fragment upstream of the AlSAP translated sequence has been isolated, cloned, and designated as the "Pr ( AlSAP )" promoter. Sequence analysis of "Pr ( AlSAP )" revealed the presence of cis-regulatory elements which could be required for abiotic stress, abscisic acid (ABA), and salicylic acid (SA) responsiveness and for tissue-specific and vascular expression. The Pr ( AlSAP ) promoter was fused to the β-glucuronidase (gusA) gene and the resulting construct transferred into tobacco. Histochemical assays of stably transformed tobacco plants showed that Pr ( AlSAP ) is active in this heterologous C3 system. While full-length gusA transcripts accumulated in whole 15, 30, and 45-day-old plants, GUS histochemical staining was only observed in leaves and stems of 45-day-old, or older, transgenic seedlings. Histological sections prepared at this stage revealed activity localized in leaf veins (phloem and bundle sheath) and stems (phloem and cortex) but not in roots. Furthermore, gusA transcripts accumulated in an age-dependent manner with a basipetal pattern in leaf and stem tissues throughout the plant. In flowers, GUS expression was detected in sepals only. The accumulation of gusA transcripts was up-regulated by salt, dehydration, ABA, and SA treatment. Altogether, these results show that, when used in a heterologous dicot system, Pr ( AlSAP ) is an age-dependent, abiotic-stress-inducible, organ-specific and tissue-specific promoter.

It is not known to what degree aquaporin-facilitated water uptake differs between root developmental regions and types of root. The aim of this study was to measure aquaporin-dependent water flow in the main types of root and root developmental regions of 14- to 17-d-old barley plants and to identify candidate aquaporins which mediate this flow. Water flow at root level was related to flow at cell and plant level. Plants were grown hydroponically. Hydraulic conductivity of cells and roots was determined with a pressure probe and through exudation, respectively, and whole-plant water flow (transpiration) determined gravimetrically in response to the commonly used aquaporin inhibitor HgCl(2). Expression of aquaporins was analysed by real-time PCR and in situ hybridization. Hydraulic conductivity of cortical cells in seminal roots was largest in lateral roots; it was smallest in the fully mature zone and intermediate in the not fully mature 'transition' zone along the main root axis. Adventitious roots displayed an even higher (3- to 4-fold) cortical cell hydraulic conductivity in the transition zone. This coincided with 3- to 4-fold higher expression of three aquaporins (HvPIP2;2, HvPIP2;5, HvTIP1:1). These were expressed (also) in cortical tissue. The largest inhibition of water flow (83-95%) in response to HgCl(2) was observed in cortical cells. Water flow through roots and plants was reduced less (40-74%). It is concluded that aquaporins contribute substantially to root water uptake in 14- to 17-d-old barley plants. Most water uptake occurs through lateral roots. HvPIP2;5, HvPIP2;2, and HvTIP1;1 are prime candidates to mediate water flow in cortical tissue.

Aquaporins are multifunctional membrane channels which belong to the family of major intrinsic proteins (MIPs) and are best known for their ability to facilitate the movement of water. In the present study, earlier results from microarray experiments were followed up. These experiments had suggested that, in barley (Hordeum vulgare L.), aquaporin family members are expressed in distinct patterns during leaf development. Real-time PCR and in situ hybridization were used to analyse the level and tissue-distribution of expression of candidate aquaporins, focusing on plasma membrane and tonoplast intrinsic proteins (PIPs, TIPs). Water channel function of seven aquaporins, whose transcripts were the most abundant and the most variable, was tested through expression in yeast and, in part, through expression in oocytes. All PIP1 and PIP2 subfamily members changed in expression during leaf development, with expression being much higher or lower in growing compared with mature tissue. The same applied to those TIPs which were expressed at detectable levels. Specific roles during leaf development are proposed for particular aquaporins.

Background: Following genome sequencing of crop plants, one of the main challenges today is determining the function of all the predicted genes. When gene validation approaches are used for woody species, the main obstacle is the low recovery rate of transgenic plants from elite or commercial cultivars. Embryogenic calli have frequently been the target tissue for transformation, but the difficulty in producing or maintaining embryogenic tissues is one of the main problems encountered in genetic transformation of many woody plants, including Coffea arabica., Results: We identified the conditions required for successful long-term proliferation of embryogenic cultures in C. arabica and designed a highly efficient and reliable Agrobacterium tumefaciens-mediated transformation method based on these conditions. The transformation protocol with LBA1119 harboring pBin 35S GFP was established by evaluating the effect of different parameters on transformation efficiency by GFP detection. Using embryogenic callus cultures, co-cultivation with LBA1119 OD600 = 0.6 for five days at 20 °C enabled reproducible transformation. The maintenance conditions for the embryogenic callus cultures, particularly a high auxin to cytokinin ratio, the age of the culture (optimum for 7-10 months of proliferation) and the use of a yellow callus phenotype, were the most important factors for achieving highly efficient transformation (> 90%). At the histological level, successful transformation was related to the number of proembryogenic masses present. All the selected plants were proved to be transformed by PCR and Southern blot hybridization., Conclusion: Most progress in increasing transformation efficiency in coffee has been achieved by optimizing the production conditions of embryogenic cultures used as target tissues for transformation. This is the first time that a strong positive effect of the age of the culture on transformation efficiency was demonstrated. Our results make Agrobacterium-mediated transformation of embryogenic cultures a viable and useful tool both for coffee breeding and for the functional analysis of agronomically important genes.

The study of arbuscular mycorrhiza often requires the staining of fungal structures using specific dyes. Fluorescent dyes such as acid fuchsin and wheat germ agglutinin conjugates give excellent results, but these compounds are either hazardous or very expensive. Here, we show that a safer and inexpensive dye, Uvitex2B, can be efficiently used to stain intraradical fungal structures formed by the arbuscular mycorrhizal fungus Glomus intraradices in three plant species: carrot, Casuarina equisetifolia, and Medicago truncatula. The intensity and stability of Uvitex2B allow the acquisition of high-quality images using not only confocal laser scanning microscopy but also epifluorescence microscopy coupled with image deconvolution. Furthermore, we demonstrate that Uvitex2B and β-glucuronidase staining are compatible and can thus be used to reveal arbuscular mycorrhizal structures in the context of promoter activation analysis.

Here we report on the characterization of rice osa-miR827 and its two target genes, OsSPX-MFS1 and OsSPX-MFS2, which encode SPX-MFS proteins predicted to be implicated in phosphate (Pi) sensing or transport. We first show by Northern blot analysis that osa-miR827 is strongly induced by Pi starvation in both shoots and roots. Hybridization of osa-miR827 in situ confirms its strong induction by Pi starvation, with signals concentrated in mesophyll, epidermis and ground tissues of roots. In parallel, we analyzed the responses of the two OsSPX-MFS1 and OsSPX-MFS2 gene targets to Pi starvation. OsSPX-MFS1 mRNA is mainly expressed in shoots under sufficient Pi supply while its expression is reduced on Pi starvation, revealing a direct relationship between induction of osa-miR827 and down-regulation of OsSPX-MFS1. In contrast, OsSPX-MFS2 responds in a diametrically opposed manner to Pi starvation. The accumulation of OsSPX-MFS2 mRNA is dramatically enhanced under Pi starvation, suggesting the involvement of complex regulation of osa-miR827 and its two target genes. We further produced transgenic rice lines overexpressing osa-miR827 and T-DNA knockout mutant lines in which the expression of osa-miR827 is abolished. Compared with wild-type controls, both target mRNAs exhibit similar changes, their expression being reduced and increased in overexpressing and knockout lines, respectively. This suggests that OsSPX-MFS1 and OsSPX-MFS2 are both negatively regulated by osa-miR827 abundance although they respond differently to external Pi conditions. We propose that this is a complex mechanism comprising fine tuning of spatial or temporal regulation of both targets by osa-miR827.

Actinorhizal symbioses are mutualistic interactions between plants and the soil bacteria Frankia that lead to the formation of nitrogen-fixing root nodules. Little is known about the signaling mechanisms controlling the different steps of the establishment of the symbiosis. The plant hormone auxin has been suggested to play a role. Here we report that auxin accumulates within Frankia-infected cells in actinorhizal nodules of Casuarina glauca. Using a combination of computational modeling and experimental approaches, we establish that this localized auxin accumulation is driven by the cell-specific expression of auxin transporters and by Frankia auxin biosynthesis in planta. Our results indicate that the plant actively restricts auxin accumulation to Frankia-infected cells during the symbiotic interaction.

The micropropagation of coconut palm has progressed rapidly; yet, there are constraints with regard to the number of somatic embryos formed and their germination. To overcome these, we tested the effect of gibberellic acid and characterized genes of the KNOX family. Gibberellic acid at 0.5 muM increased 1.5-fold the number of calli forming somatic embryos and twofold the number of somatic embryos per callus, calli with germinating embryos and the number of germinating somatic embryos per callus. With regard to the study of KNOX family genes, the complete sequences of two KNOX-like genes were obtained for CnKNOX1 and CnKNOX2. The deduced amino acid sequence of both showed highly conserved domains characteristic of KNOX genes. CnKNOX1 showed high homology with KNOX class I proteins. CnKNOX1 expression was detected throughout the embryogenesis process except in somatic embryos at the pro-globular stage, and was highest in somatic embryos at the coleoptilar stage. No detection of CnKNOX1 expression occurred in calli with aberrant embryos. The addition of gibberellic acid stimulated the expression of CnKNOX1 earlier and the relative expression at all stages was higher. CnKNOX2 expression occurred at all stages peaking at the globular stage, but gibberellic acid treatment decreased the expression. Gene expression was also analyzed in tissues of different organs of adult palms. With CnKNOX1, high level of expression was found in tissues of organs with, but not in those without, meristem, whereas CnKNOX2 expression was detected in tissues with and also in those without meristem.

Background: Despite the wide spread application of confocal and multiphoton laser scanning microscopy in plant biology, leaf phenotype assessment still relies on two-dimensional imaging with a limited appreciation of the cells' structural context and an inherent inaccuracy of cell measurements. Here, a successful procedure for the three-dimensional imaging and analysis of plant leaves is presented., Results: The procedure was developed based on a range of developmental stages, from leaf initiation to senescence, of soil-grown Arabidopsis thaliana (L.) Heynh. Rigorous clearing of tissues, made possible by enhanced leaf permeability to clearing agents, allowed the optical sectioning of the entire leaf thickness by both confocal and multiphoton microscopy. The superior image quality, in resolution and contrast, obtained by the latter technique enabled the three-dimensional visualisation of leaf morphology at the individual cell level, cell segmentation and the construction of structural models. Image analysis macros were developed to measure leaf thickness and tissue proportions, as well as to determine for the epidermis and all layers of mesophyll tissue, cell density, volume, length and width. For mesophyll tissue, the proportion of intercellular spaces and the surface areas of cells were also estimated. The performance of the procedure was demonstrated for the expanding 6th leaf of the Arabidopsis rosette. Furthermore, it was proven to be effective for leaves of another dicotyledon, apple (Malus domestica Borkh.), which has a very different cellular organisation., Conclusions: The pipeline for the three-dimensional imaging and analysis of plant leaves provides the means to include variables on internal tissues in leaf growth studies and the assessment of leaf phenotypes. It also allows the visualisation and quantification of alterations in leaf structure alongside changes in leaf functioning observed under environmental constraints. Data obtained using this procedure can further be integrated in leaf development and functioning models.

Quantitative information on growing organs is required to better understand morphogenesis in both plants and animals. However, detailed analyses of growth patterns at cellular resolution have remained elusive. We developed an approach, multiangle image acquisition, three-dimensional reconstruction and cell segmentation-automated lineage tracking (MARS-ALT), in which we imaged whole organs from multiple angles, computationally merged and segmented these images to provide accurate cell identification in three dimensions and automatically tracked cell lineages through multiple rounds of cell division during development. Using these methods, we quantitatively analyzed Arabidopsis thaliana flower development at cell resolution, which revealed differential growth patterns of key regions during early stages of floral morphogenesis. Lastly, using rice roots, we demonstrated that this approach is both generic and scalable.

Grapevine (Vitis vinifera), the genome sequence of which has recently been reported, is considered as a model species to study fleshy fruit development and acid fruit physiology. Grape berry acidity is quantitatively and qualitatively affected upon increased K(+) accumulation, resulting in deleterious effects on fruit (and wine) quality. Aiming at identifying molecular determinants of K(+) transport in grapevine, we have identified a K(+) channel, named VvK1.1, from the Shaker family. In silico analyses indicated that VvK1.1 is the grapevine counterpart of the Arabidopsis AKT1 channel, known to dominate the plasma membrane inward conductance to K(+) in root periphery cells, and to play a major role in K(+) uptake from the soil solution. VvK1.1 shares common functional properties with AKT1, such as inward rectification (resulting from voltage sensitivity) or regulation by calcineurin B-like (CBL)-interacting protein kinase (CIPK) and Ca(2+)-sensing CBL partners (shown upon heterologous expression in Xenopus oocytes). It also displays distinctive features such as activation at much more negative membrane voltages or expression strongly sensitive to drought stress and ABA (upregulation in aerial parts, downregulation in roots). In roots, VvK1.1 is mainly expressed in cortical cells, like AKT1. In aerial parts, VvK1.1 transcripts were detected in most organs, with expression levels being the highest in the berries. VvK1.1 expression in the berry is localized in the phloem vasculature and pip teguments, and displays strong upregulation upon drought stress, by about 10-fold.VvK1.1 could thus play a major role in K(+) loading into berry tissues, especially upon drought stress.