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Cryopreservation has several advantages over other ex situ conservation methods, and indeed is the only viable storage method for the long term conservation of most plant species. However, despite many advances in this field, it is increasingly clear that some species are ill-equipped to overcome the intense stress imposed by the cryopreservation process, making protocol development incredibly difficult using traditional trial and error methods. Cryobiotechnology approaches have been recently recognised as a strategic way forward, utilising intimate understanding of biological systems to inform development of more effective cryopreservation protocols. Mitochondrial function is a model candidate for a cryobiotechnological approach, as it underpins not only energy provision, but also several other key determinants of germplasm outcome, including stress response, reduction-oxidation status, and programmed cell death. Extensive research in animal cell and tissue cryopreservation has established a clear link between mitochondrial health and cryopreservation survival, but also indicates that mitochondria are routinely subject to damage from multiple aspects of the cryopreservation process. Evidence is already emerging that mitochondrial dysfunction may also occur in plant cryopreservation, and this research can be greatly expanded by using considered applications of innovative technologies. A range of mitochondria-targeted prophylactic and therapeutic interventions already exist with potential to improve cryopreservation outcomes through mitochondrial function., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Background: Both underground rhizomes/buds and above-ground Moso bamboo (Phyllostachys heterocycla) shoots/culms/branches are connected together into a close inter-connecting system in which nutrients are transported and shared among each organ. However, the starch storage and utilization mechanisms during bamboo shoot growth remain unclear. This study aimed to reveal in which organs starch was stored, how carbohydrates were transformed among each organ, and how the expression of key genes was regulated during bamboo shoot growth and developmental stages which should lay a foundation for developing new theoretical techniques for bamboo cultivation., Results: Based on changes of the NSC content, starch metabolism-related enzyme activity and gene expression from S0 to S3, we observed that starch grains were mainly elliptical in shape and proliferated through budding and constriction. Content of both soluble sugar and starch in bamboo shoot peaked at S0, in which the former decreased gradually, and the latter initially decreased and then increased as shoots grew. Starch synthesis-related enzymes (AGPase, GBSS and SBE) and starch hydrolase (α-amylase and β-amylase) activities exhibited the same dynamic change patterns as those of the starch content. From S0 to S3, the activity of starch synthesis-related enzyme and starch amylase in bamboo rhizome was significantly higher than that in bamboo shoot, while the NSC content in rhizomes was obviously lower than that in bamboo shoots. It was revealed by the comparative transcriptome analysis that the expression of starch synthesis-related enzyme-encoding genes were increased at S0, but reduced thereafter, with almost the same dynamic change tendency as the starch content and metabolism-related enzymes, especially during S0 and S1. It was revealed by the gene interaction analysis that AGPase and SBE were core genes for the starch and sucrose metabolism pathway., Conclusions: Bamboo shoots were the main organ in which starch was stored, while bamboo rhizome should be mainly functioned as a carbohydrate transportation channel and the second carbohydrate sink. Starch metabolism-related genes were expressed at the transcriptional level during underground growth, but at the post-transcriptional level during above-ground growth. It may be possible to enhance edible bamboo shoot quality for an alternative starch source through genetic engineering., (© 2021. The Author(s).)

Epigenetic modifications within promoter sequences can act as regulators of gene expression. Shoot regeneration is influenced by both DNA methylation and histone methylation, but the mechanistic basis of this regulation is obscure. Here, we identified 218 genes related to the regeneration capacity of callus that were differentially transcribed between regenerable calli (RC) and non-regenerable calli (NRC) in Arabidopsis thaliana. An analysis of the promoters of five of the differentially expressed genes (FWA, ACC1, TFL1, MAX3, and GRP3) pointed to an inverse relationship between cytosine methylation and transcription. The FWA promoter was demethylated and highly expressed in NRC, whereas it was methylated and expressed at low levels in RC. Explants of the hypomethylation mutants fwa-1 and fwa-2 showed strong levels of FWA expression and regenerated less readily than the wild type, suggesting that FWA inhibits direct in vitro shoot regeneration. WUSCHEL-RELATED HOMEOBOX 9 (WOX9), which is required for shoot apical meristem formation, was directly repressed by FWA. Overexpressing WOX9 partly rescued the shoot regeneration defect of fwa-2 plants. These findings suggest that cytosine methylation of the FWA promoter forms part of the regulatory system governing callus regenerability and direct in vitro shoot regeneration., (© 2021 The Authors. Journal of Integrative Plant Biology published by John Wiley & Sons Australia, Ltd on behalf of Institute of Botany, Chinese Academy of Sciences.)

Somatic embryogenesis (SE) is the most important plant biotechnology process for plant regeneration, propagation, genetic transformation and genome editing of coffee, Coffea arabica L. Somatic embryo (SEs) conversion to plantlets is the principal bottleneck for basic and applied use of this process. In this study we focus on the maturation of SEs of C. arabica var. Typica. SEs conversion to plantlet up to 95.9% was achieved under osmotic stress, using 9 g/L gelrite, as compared with only 39.34% in non-osmotic stress. Mature SEs induced in osmotic stress developed shoot and root apical meristems, while untreated SEs were unable to do it. C. arabica regenerated plants from osmotic stress were robust, with higher leaf and root area and internode length. To understand a possible regulatory mechanism, gene expression of key genes of C. arabica, homologous to sequences in the Arabidopsis thaliana genome, were analyzed. A set of two component system and cytokinin signaling-related coding genes (AHK1, AHK3, AHP4 and ARR1) which interact with WUSCHEL and WOX5 homedomains and morphogenic genes, BABY-BOOM, LEC1, FUS3 and AGL15, underwent significant changes during maturation of SEs of C. arabica var. Typica. This protocol is currently being applied in genetic transformation with high rate of success.

Cucurbit phloem is complex, with large sieve tubes on both sides of the xylem (bicollateral phloem), and extrafascicular elements that form an intricate web linking the rest of the vasculature. Little is known of the physical interconnections between these networks or their functional specialization, largely because the extrafascicular phloem strands branch and turn at irregular angles. Here, export in the phloem from specific regions of the lamina of cucumber (Cucumis sativus L.) was mapped using carboxyfluorescein and 14 C as mobile tracers. We also mapped vascular architecture by conventional microscopy and X-ray computed tomography using optimized whole-tissue staining procedures. Differential gene expression in the internal (IP) and external phloem (EP) was analyzed by laser-capture microdissection followed by RNA-sequencing. The vascular bundles of the lamina form a nexus at the petiole junction, emerging in a predictable pattern, each bundle conducting photoassimilate from a specific region of the blade. The vascular bundles of the stem interconnect at the node, facilitating lateral transport around the stem. Elements of the extrafascicular phloem traverse the stem and petiole obliquely, joining the IP and EP of adjacent bundles. Using pairwise comparisons and weighted gene coexpression network analysis, we found differences in gene expression patterns between the petiole and stem and between IP and EP, and we identified hub genes of tissue-specific modules. Genes related to transport were expressed primarily in the EP while those involved in cell differentiation and development as well as amino acid transport and metabolism were expressed mainly in the IP., (© 2021 Society for Experimental Biology and John Wiley & Sons Ltd.)

Dodder is a holoparasitic flowering plant that re-establishes parasitism with the host when broken off from the host. However, how in vitro dodder shoots recycle stored nutrients to maintain growth for reparasitizing hosts is not well characterized. Here, the spatial and temporal distribution characteristics of carbohydrates and reactive oxygen species (ROS) were analysed to explore the mechanism of recycling stored nutrients in dodder shoots in vitro. Our results showed that in vitro dodder shoots grew actively for more than 10 d, while dry mass decreased continuously. During this process, the transcript levels and activities of amylases gradually increased until 2 d and then declined in basal stems, which induced starch degradation at the tissue, cellular and subcellular levels. Additionally, the distribution characteristics of H 2 O 2 and the activities and transcript levels of antioxidant enzymes indicated that shoot tips exhibited more robust ROS-scavenging capacity, and basal stems maintained higher ROS accumulation. Comparative proteomics analysis revealed that starch in basal stems acted as an energy source, and the glycolysis, TCA cycle and pentose phosphate pathway represented the energy supply for shoot tip elongation with time. These results indicated that efficient nutrient recycling and ROS modulation facilitated the parasitism of dodder grown in vitro by promoting shoot elongation growth to reach the host., (Copyright © 2020 Elsevier B.V. All rights reserved.)

In the present research, Silene vulgaris as a representative species growing on both unpolluted and heavy metal (HM) polluted terrains were used to identify ecotype-specific responses to metallic stress. Growth, cell ultrastructure and element accumulations were compared between non-metallicolous (NM), calamine (CAL) and serpentine (SER) specimens untreated with HMs and treated with Pb, Cd and Zn ions under in vitro conditions. Moreover, proteins' modifications related to their level, carbonylation and degradations via vacuolar proteases were verified and linked with potential mechanisms to cope with ions toxicity. Our experiment revealed diversified strategy of HM uptake in NM and both metallicolous ecotypes, in which antagonistic relationship of Zn and Pb/Cd ions provided survival benefits for the whole organism. Despite this similarity, growth rate and metabolic pathways induced in CAL and SER shoots varied significantly. Exposition to HMs in CAL culture led to drop in protein level by approximately 16% compared to the control. This parameter nearly correlated with the enhanced activity of proteases at pH 5.2 as well as possible glutamate changes to proline and reduced glutathione, resulting in intensified growth and first signs of cell senescence. In turn, SER shoots were characterized by growth retardation (to 53% of the control), although protein level and carbonylation were not modified, while a deeper insight into protein network showed its remodeling towards production of polyamines and 2-oxoglutarate delivered to the Krebs cycle. Contrary, an uncontrolled HM influx in NM shoots contributed to morpho-structural disorders accompanied by an increase activity of proteases involved in the degradation of oxidized proteins, what pointed to metal-induced autophagy. Taken together, S. vulgaris ecotypes respond to stress by triggering various mechanisms engaged their survival and/or death under HM treatment., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Silver nanoparticles (AgNPs) are presently the most commonly generated engineered nanomaterials and are found in a wide range of agro-commercial products. The present study was designed to synthesize AgNPs biologically using Ochradenus arabicus leaves and investigate their effect on the morphophysiological properties of Maerua oblongifolia raised in vitro. Physicochemical methods (ultraviolet-visible spectroscopy, Fourier transform infrared spectroscopy, and transmission electron microscopy were performed for characterization and for obtaining microphotographs of the AgNPs. Shoots of M. oblongifolia (2-3 cm) grown in Murashige and Skoog medium supplemented with different concentrations of AgNPs (0, 10, 20, 30, 40, or 50 mg L -1 ) were used. Following 6 weeks of in vitro shoot regeneration, the shoot number, shoot length, leaf number, fresh weight, dry weight, chlorophyll content, total protein, proline level, and antioxidant enzyme activities of the plants were quantified. We found that 20 mg L -1 AgNPs increased the shoot number, shoot length, fresh weight, dry weight, and chlorophyll content of the plants. The maximum total protein was recorded in plants that were administered the lowest dose of AgNPs (10 mg L -1 ), while high concentrations of AgNPs (40 and 50 mg L -1 ) increased the levels of proline and the enzymes superoxide dismutase and catalase. Our results indicate that green-synthesized AgNPs may be of agricultural and medicinal interest owing to their effects on plants in vitro.

The impact of nanotechnology in the field of agricultural sciences creates the need to study in greater detail the effect of products offering nanoparticles for application in plant species of agricultural interest. The objective of this study was to determine the response of stevia (Stevia rebaudiana B.) in vitro to different concentrations of AgNPs (silver nanoparticles), as well as to characterize and identify their absorption, translocation and accumulation mechanisms. Nodal segments of stevia grown in MS medium supplemented with AgNPs (0,12.5, 25, 50,100 and 200 mg L -1 ) were used. After 30 days of in vitro shoot proliferation, the number of shoots per explant, shoot length, chlorophyll content, dry matter content and the metallic silver (Ag) content of the plants were quantified. In addition, characterization, transport and accumulation of silver nanoparticles were performed by microscopic analysis. AgNPs were shown to be present in epidermal stem cells, within vascular bundles and in intermembrane spaces. In leaves, they were observed in ribs and stomata. The current and future use of AgNPs in agricultural sciences opens up the possibility of studying their effects on different plant species.

Shoot meristems, which harbor a small population of stem cells, are responsible for generating new above-ground organs in plants. The proliferation and differentiation of these stem cells is regulated by a genetic pathway involving two key meristematic genes: CLAVATA3 ( CLV3 ) and WUSCHEL ( WUS ). However, it is not well understood how CLV3 and WUS expression domains in the shoot meristems are specified and maintained during post-embryogenic development. Here, we show that a tomato mutant with fasciated stems, flowers and fruits, due to impaired stem cell activity, is defective in a LITTLE ZIPPER gene denoted as DEFECTIVE TOMATO MERISTEM ( DTM ). DTM forms a negative feedback loop with class III homeodomain-leucine zipper (HD-ZIP III) transcription factors to confine CLV3 and WUS expression to specific domains of the shoot meristems. Our findings reveal a new layer of complexity in the regulation of plant stem cell homeostasis., Competing Interests: The authors declare no competing interests.

Confocal microscopy is widely used to live-image plant tissue. Cell outlines can be visualized using fluorescent probes that mark the cell wall or plasma membrane, enabling the confocal microscope to be used as a 3D scanner with submicron precision. After imaging, the data needs to be analyzed by specialized software to quantify the features of interest, such as cell size and shape, growth rates and anisotropy, and gene expression. Here we present a protocol for the 3D image processing software MorphoGraphX ( www.MorphoGraphX.org ) using time-lapse images of an Arabidopsis thaliana sepal and the shoot apex of tomato.

Complex geometry of plant organs and various types of organ surface deformation, including growth or hygroscopic movements, can be analyzed using sequential replica method. It enables obtaining a time-lapse series of high resolution images visualizing details of the examined surface and provides data sufficient for detailed computation of parameters characterizing surface deformation and geometry. Series of molds, made in dental polymer, representing the examined surface are used to obtain casts in epoxy resin or nail polish replicas, which are ready for microscopic examination, while the structure itself remains intact. Images obtained from the epoxy casts in scanning electron microscopy can be further used for 3D reconstruction and computation of local geometry. The sequential replica method is a universal method and can be applied to image complex shapes of a range of structures, like meristems, flowers, leaves, scarious bracts, or trichomes. Different plant species growing in various conditions can be studied.

Analysis of meristem shape and gene expression pattern has been conducted in many species over the past decades. Recent live imaging techniques have allowed for an unprecedented accumulation of data on the biology of meristematic cells, as well as a better understanding of the molecular and biophysical mechanisms behind shape changes in this tissue. Here we describe in detail how to prepare shoot apices of both Arabidopsis and tomato, in order to image them over time using a confocal microscope equipped with a long distance water-dipping lens.

Induction of somatic embryogenesis and complete plantlet regeneration from callus culture of Hibiscus sabdariffa L. var. HS4288 has been made. Leaf and root explants were cultured on Murashige and Skoog (MS) and Driver-Kuniyuki Walnut (DKW) basal media supplemented with different concentrations of synthetic auxins and cytokinins. Root explants on DKW medium supplemented with 2.26μM 2, 4-Dichlorophenoxyacetic acid (2, 4-D) and 4.65μM kinetin (KIN) induced highest percentage (70%) of embryogenic calli. Average number of globular embryos per root derived callus produced within 6 weeks of culture initiation on MS media with different plant growth regulators (PGRs) ranged from 2.27±0.12 to 8.80±0.17 and that of cotyledonary embryos ranged from 0.00 to 2.53±0.20. On DKW medium comparatively more globular embryos (2.70±0.15 to 14.53±0.23) and cotyledonary embryos (0.00 to 8.90±0.17) were produced than that of MS medium. Regeneration of complete plantlets was highest (76.67%) when embryogenic calli with mature somatic embryos were grown on DKW medium containing 2.32μM KIN and 2.22μM 6-Benzyladenine (BA). Plants were primarily hardened in humidity, temperature and light controlled chamber and finally in a greenhouse showed 70% survival ability. Different stages of somatic embryogenesis process in the root derived embryogenic calli were elaborated in detail by morphological, histological and SEM study. The data were statistically analyzed by Duncan Multiple range test (p ≤ 0.05) and Principal component analysis (PCA). Flow cytometry and Inter-simple sequence repeats (ISSR) marker analysis confirmed that there was no genetic variation within the regenerated plants., Competing Interests: The authors have declared that no competing interests exist.

Background and Aims: Setaria viridis is being promoted as a model C4 photosynthetic plant because it has a small genome (~515 Mb), a short life cycle (~60 d) and it can be transformed. Unlike other C4 grasses such as maize, however, there is very little information about how C4 leaf anatomy (Kranz anatomy) develops in S. viridis. As a foundation for future developmental genetic studies, we provide an anatomical and ultrastructural framework of early shoot development in S. viridis, focusing on the initiation of Kranz anatomy in seed leaves., Methods: Setaria viridis seeds were germinated and divided into five stages covering development from the dry seed (stage S0) to 36 h after germination (stage S4). Material at each of these stages was examined using conventional light, scanning and transmission electron microscopy., Key Results: Dry seeds contained three embryonic leaf primordia at different developmental stages (plastochron 1-3 primordia). The oldest (P3) leaf primordium possessed several procambial centres whereas P2 displayed only ground meristem. At the tip of P3 primordia at stage S4, C4 leaf anatomy typical of the malate dehydrogenase-dependent nicotinamide dinucleotide phosphate (NADP-ME) subtype was evident in that vascular bundles lacked a mestome layer and were surrounded by a single layer of bundle sheath cells that contained large, centrifugally located chloroplasts. Two to three mesophyll cells separated adjacent vascular bundles and one mesophyll cell layer on each of the abaxial and adaxial sides delimited vascular bundles from the epidermis., Conclusions: The morphological trajectory reported here provides a foundation for studies of gene regulation during early leaf development in S. viridis and a framework for comparative analyses with other C4 grasses.

Pigeonpea (Cajanus cajan), a tropical grain legume with low input requirements, is expected to continue to have an important role in supplying food and nutritional security in developing countries in Asia, Africa and the tropical Americas. From whole-genome resequencing of 292 Cajanus accessions encompassing breeding lines, landraces and wild species, we characterize genome-wide variation. On the basis of a scan for selective sweeps, we find several genomic regions that were likely targets of domestication and breeding. Using genome-wide association analysis, we identify associations between several candidate genes and agronomically important traits. Candidate genes for these traits in pigeonpea have sequence similarity to genes functionally characterized in other plants for flowering time control, seed development and pod dehiscence. Our findings will allow acceleration of genetic gains for key traits to improve yield and sustainability in pigeonpea.

This study investigates the histology and subcellular features of secretory cavities during the development of the shoot apex of Metrodorea nigra A. St.-Hil. in order to better understand the functioning of these glands. This Rutaceae species is a very suitable model for studying secretory cavity life span, since the shoot apex exhibits both dormant and growth stages during its annual cycle. Shoot apices were collected during the dormant and growth stages from populations of M. nigra growing under natural conditions. Materials were processed using standard techniques for light and electron microscopy. The secretory cavities originate under the protodermis, and their initiation is restricted to the early developmental stage of shoot organs, which are protected by a hood-shaped structure. Secretory cavities have a multi-seriate epithelium surrounding a lumen that expands schizolysigenously. Oil production begins before lumen formation. When the shoot apex resumes development after the dormant stage, the glands remain active in oil secretion in the developing shoot apex and fully expanded leaves. The mature epithelial cells are flattened and exhibit very thin walls, large oil bodies, leucoplasts surrounded by endoplasmic reticulum, and mitochondria with unusual morphology. The tangential walls of the epithelial cells facing the lumen undergo continuous peeling. The vacuole extrusion appears to be the primary mode of release oil into the lumen, in an exocytotic way. The continuity of oil secretion is ensured by the replacement of the damaged inner epithelial cells by divisions in the parenchyma layer that surround the oil gland, likely a meristematic sheath.

The primary thickening growth of Moso (Phyllostachys edulis) underground shoots largely determines the culm circumference. However, its developmental mechanisms remain largely unknown. Using an integrated anatomy, mathematics and genomics approach, we systematically studied cellular and molecular mechanisms underlying the growth of Moso underground shoots. We discovered that the growth displayed a spiral pattern and pith played an important role in promoting the primary thickening process of Moso underground shoots and driving the evolution of culms with different sizes among different bamboo species. Different with model plants, the shoot apical meristem (SAM) of Moso is composed of six layers of cells. Comparative transcriptome analysis identified a large number of genes related to the vascular tissue formation that were significantly upregulated in a thick wall variant with narrow pith cavity, mildly spiral growth, and flat and enlarged SAM, including those related to plant hormones and those involved in cell wall development. These results provide a systematic perspective on the primary thickening growth of Moso underground shoots, and support a plausible mechanism resulting in the narrow pith cavity, weak spiral growth but increased vascular bundle of the thick wall Moso., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

Premise of the Study: Fibonacci phyllotactic patterns in seed plants are well documented, but whether such predominance holds true for lower vascular plants is relatively unknown. We investigated Diphasiastrum digitatum (Lycopodiaceae) phyllotaxis throughout its ontogeny to extend our knowledge of pattern frequency of lower vascular plants and to measure quantitative variables associated with discontinuous phyllotactic transitions. These investigations allowed us to test whether the same mechanisms inherent in shoot apical meristem (SAM) development of seed plants are applicable to early-diverged lower vascular plants SAM development., Methods: Divergence angle, plastochron ratio, leaf insertion angle, circumferential ratio, radial ratio, half conic angle, area, circumference, and circularity of the shoot apical meristem were compared among different phyllotactic patterns and different meristem types observed throughout D. digitatum ontogeny, using scanning electron microscopy., Key Results: Fibonacci patterns were not predominant during six stages of D. digitatum ontogeny. In all five cases of discontinuous transition associated with strobili formation, divergence angle was the only variable that has changed consistently., Conclusions: The predominance of non-Fibonacci patterns/series in D. digitatum is inconsistent with the prediction of interpretive model of phyllotaxis. We hypothesize this is because its SAM, due to its frequent dichotomy, is not circular and primordia initiation is restricted spatially and temporally at the beginning of pattern formation. Change in divergence angle associated with discontinuous transitions is most likely due to the change of the location of new auxin maxima, due to the change of SAM shape and size., (© 2017 Botanical Society of America.)

Recent advances in confocal microscopy, coupled with the development of numerous fluorescent reporters, provide us with a powerful tool to study the development of plants. Live confocal imaging has been used extensively to further our understanding of the mechanisms underlying the formation of roots, shoots and leaves. However, it has not been widely applied to flowers, partly because of specific challenges associated with the imaging of flower buds. Here, we describe how to prepare and grow shoot apices of Arabidopsis in vitro, to perform both single-point and time-lapse imaging of live, developing flower buds with either an upright or an inverted confocal microscope., (Copyright © 2016 Elsevier Inc. All rights reserved.)

Colleters are secretory structure present on many families including Rubiaceae. Particular characteristics have been described about colleters secretory cells, however senescence process are still under debate. Tocoyena bullata (Vell.) Mart. (Rubiaceae) shoot apex were collected at Jardim Botânico do Rio de Janeiro, RJ/Brazil. Stipules were separated and fragments were fixed in 2.5% glutaraldehyde and 4.0% formaldehyde in 0.05 m sodium cacodylate buffer, pH 7.2, post fixed in 1.0% osmium tetroxide in the same buffer, dehydrated in acetone, critical-point-drying, sputtered coated and observed. For light microscopy fragments were fixed and dehydrated, infiltrated with historesin and stained with 1% toluidine blue. For transmission electron microscopy, the samples were infiltrated with Epoxi resin. Colleters are present on stipule adaxial surface. On the beginning of development, these structures are recognized as small projections. Later on, colleters differentiated and secrete by cuticle rupture. The colleters senescence occurs in a concomitant and indissoluble way of programmed cell death. Ultrastructural analyses during the process strongly suggest the senescence is based on a non-autolitic programmed cell death. T. bullata colleters, present at stipule abaxial surface are cylindrical secretory structures. Colleters secretory cells originated as stipule projections; differentiate; secrete and senesce by programmed cell death. The secretion and the cell dead occurs in a concomitantly and indissoluble way., (© 2016 German Botanical Society and The Royal Botanical Society of the Netherlands.)

The ability of plants to match their reproductive output with favorable environmental conditions has major consequences both for lifetime fitness and geographic patterns of diversity. In temperate ecosystems, some plant species have evolved the ability to use winter nonfreezing cold (vernalization) as a cue to ready them for spring flowering. However, it is unknown how important the evolution of vernalization responsiveness has been for the colonization and subsequent diversification of taxa within the northern and southern temperate zones. Grasses of subfamily Pooideae, including several important crops, such as wheat (Triticum aestivum), barley (Hordeum vulgare), and oats (Avena sativa), predominate in the northern temperate zone, and it is hypothesized that their radiation was facilitated by the early evolution of vernalization responsiveness. Predictions of this early origin hypothesis are that a response to vernalization is widespread within the subfamily and that the genetic basis of this trait is conserved. To test these predictions, we determined and reconstructed vernalization responsiveness across Pooideae and compared expression of wheat vernalization gene orthologs VERNALIZATION1 (VRN1) and VRN3 in phylogenetically representative taxa under cold and control conditions. Our results demonstrate that vernalization responsive Pooideae species are widespread, suggesting that this trait evolved early in the lineage and that at least part of the vernalization gene network is conserved throughout the subfamily. These results are consistent with the hypothesis that the evolution of vernalization responsiveness was important for the initial transition of Pooideae out of the tropics and into the temperate zone., (© 2016 American Society of Plant Biologists. All rights reserved.)

Dendrobium sonia-28 is a popular orchid hybrid due to its flowering recurrence and dense inflorescences. Unfortunately, it is being decimated by fungal diseases, especially those caused by Fusarium proliferatum. In this study, selection of F. proliferatum-tolerant protocorm-like bodies (PLBs) was carried out by assessing the effects of differing concentrations of fusaric acid (FA). PLBs were cultured on Murashige and Skoog (MS) medium supplemented with 0.05 to 0.2 millimolar (mM) concentrations of FA. Higher concentrations of FA increased mortality of PLBs and reduced their growth. The survival rate for 0.05 mM FA was 20 % but only 1 % at the highest dose of 0.2 mM. Additionally, two different size ranges of PLBs were investigated, and growth increased more at lower FA concentrations for larger PLBs, whilst the growth rate of smaller PLBs was inhibited at an FA concentration of 0.2 mM. Histological examination using transmission electron microscopy (TEM) and scanning electron microscopy (SEM) analyses disclosed severe cell wall and organelle damage, as well as stomatal closure in PLBs treated with the high FA concentrations. Reductions in plantlet growth were much greater at the highest concentrations of FA. Some randomly amplified polymorphic DNA (RAPD) markers clearly discriminated between selected and non-selected variants of Dendrobium sonia-28, showing different banding patterns for each FA concentration and specific bands for selected and control plants.

Chrysanthemum morifolium 'Borami' and 'Secret Pink' showing symptoms of stunt disease caused by chrysanthemum stunt viroid (CSVd) and 'Yellow Cap' showing chlorotic mottle disease caused by chrysanthemum chlorotic mottle viroid (CChMVd) were confirmed to be infected by the respective viroids by using reverse transcription polymerase chain reaction (RT-PCR). Real-time PCR results showed that the viroid concentrations in the infected cultivars varied between the different regions of origin (Chilgok, Gumi, and Gyeongsan). We applied a cryopreservation protocol for elimination of CSVd from naturally infected 'Borami' collected from Gumi, showing the lowest concentration of CSVd, by varying several factors such as plant vitrification solutions (PVS2 and PVS3), duration of exposure to liquid nitrogen, shoot-tip size, and low-temperature treatment. The solution (PVS2) and low-temperature treatment were found to be critical factors determining the efficacy of viroid elimination. We optimized the protocol by combining of all resulted optimal factors and tested the applicability of the protocol in 'Borami' collected from Chilgok and Gyeongsan and in 'Secret Pink' from Chilgok, Gumi, and Gyeongsan, which displayed different viroid concentrations. We found that the elimination rates varied depending on the cultivar and region of origin. Similar results were observed when the protocol was applied to eliminate CChMVd from the 'Yellow Cap' collected from the same regions. Finally, we found that nested PCR is more reliable for viroid detection than RT-PCR. Overall, cryopreservation can be used to eliminate viroids from infected chrysanthemums; however, the efficacy depends on genotype and initial viroid concentration.

This study evaluates the responses of Lavandula dentata under drought conditions to the inoculation with single autochthonous arbuscular mycorrhizal (AM) fungus (five fungal strains) or with their mixture and the effects of these inocula with a native Bacillus thuringiensis (endophytic bacteria). These microorganisms were drought tolerant and in general, increased plant growth and nutrition. Particularly, the AM fungal mixture and B. thuringiensis maximized plant biomass and compensated drought stress as values of antioxidant activities [superoxide dismutase (SOD), catalase (CAT) and ascorbate peroxidase APX)] shown. The AMF-bacteria interactions highly reduced the plant oxidative damage of lipids [malondialdehyde (MDA)] and increased the mycorrhizal development (mainly arbuscular formation representative of symbiotic functionality). These microbial interactions explain the highest potential of dually inoculated plants to tolerate drought stress. B. thuringiensis "in vitro" under osmotic stress does not reduce its PGPB (plant growth promoting bacteria) abilities as indole acetic acid (IAA) and ACC deaminase production and phosphate solubilization indicating its capacity to improve plant growth under stress conditions. Each one of the autochthonous fungal strains maintained their particular interaction with B. thuringiensis reflecting the diversity, intrinsic abilities and inherent compatibility of these microorganisms. In general, autochthonous AM fungal species and particularly their mixture with B. thuringiensis demonstrated their potential for protecting plants against drought and helping plants to thrive in semiarid ecosystems., (Copyright © 2015 Elsevier GmbH. All rights reserved.)

An efficient protocol for micropropagation of Canna indica L., an economically and pharmaceutically important plant, was standardized using rhizome explants, excised from two-month-old aseptic seedlings. Complete plant regeneration was induced on MS medium supplemented with 3.0 mg/L BAP plus 1.5 mg/L NAA, which produced the highest number of shoots (73.3 ± 0.5%) and roots (86.7 ± 0.4%) after 2 weeks. Furthermore, the optimum media for multiple shoots regeneration were recorded on MS enriched with 7.0 mg/L BAP (33.0 ± 0.5%). Plantlets obtained were transplanted to pots after two months and acclimatized in the greenhouse, with 75% survival. In addition, ultrastructural studies showed that rhizomes of in vitro grown specimens were underdeveloped compared to the in vivo specimens, possibly due to the presence of wide spaces. Meanwhile, the leaves of in vivo specimens had more open stomata compared to in vitro specimens, yet their paracytic stomata structures were similar. Hence, there were no abnormalities or major differences between in vitro regenerants and mother plants.

Colleters are secretory structures that produce and release mucilage or a mucilage-resin mixture protecting meristems and young structures against desiccation, herbivores, and pathogens. The secretions may vary in colleters of same or different types, indicating that the functionality of colleters may be more specific than previously thought. In this study, we compared 17 Rubiaceae species from savanna and forest environment focusing on colleter secretions and its ecological role. First, we evaluated the morphology, distribution, and histochemistry of stipular colleters using light and scanning electron microscopy. Additionally, we investigated the phenology, microclimate, and the proportion of damaged apices in the savanna and forest species. We recorded standard-type colleters, variable in distribution and size, in 14 of the 17 studied species. The secretion varied from predominantly hydrophilic, mixed to predominantly lipophilic. During the budding period, secretion covered the vegetative apices. Savanna species had a prevalence of lipid secretion in habitats with higher luminosity, which had a lower proportion of damaged apices. In contrast, forest species occurred in habitats with lower luminosity and had a higher proportion of damaged apices, in general with the absence of lipids in the colleters. These results highlight that colleters with similar morphology clearly differed in secretions among species, especially between species from savanna and forest, in which the colleters appear potentially associated with protection against irradiation in savanna, but not in the forest environment.

Plant shoot branching is pivotal for developmental plasticity and crop yield. The formation of branch meristems is regulated by several key transcription factors including REGULATOR OF AXILLARY MERISTEMS1 (RAX1), RAX2, and RAX3. However, the regulatory network of shoot branching is still largely unknown. Here, we report the identification of EXCESSIVE BRANCHES1 (EXB1), which affects axillary meristem (AM) initiation and bud activity. Overexpression of EXB1 in the gain-of-function mutant exb1-D leads to severe bushy and dwarf phenotypes, which result from excessive AM initiation and elevated bud activities. EXB1 encodes the WRKY transcription factor WRKY71, which has demonstrated transactivation activities. Disruption of WRKY71/EXB1 by chimeric repressor silencing technology leads to fewer branches, indicating that EXB1 plays important roles in the control of shoot branching. We demonstrate that EXB1 controls AM initiation by positively regulating the transcription of RAX1, RAX2, and RAX3. Disruption of the RAX genes partially rescues the branching phenotype caused by EXB1 overexpression. We further show that EXB1 also regulates auxin homeostasis in control of shoot branching. Our data demonstrate that EXB1 plays pivotal roles in shoot branching by regulating both transcription of RAX genes and auxin pathways., (© 2015 American Society of Plant Biologists. All rights reserved.)

Premise of the Study: The phylogenetic position of Ceratophyllum is still controversial in recent molecular analyses of angiosperms, with various suggestions of a sister group relation to all other angiosperms, eudicots, monocots, eudicots + monocots, and magnoliids. Therefore, the morphological characters of Ceratophyllum are important for resolving the phylogeny of angiosperms. In this study, we observed the detailed developmental anatomy of all lateral organs and their configurations to elucidate the floral development and phyllotactic pattern of Ceratophyllum demersum., Methods: We observed fixed shoots of C. demersum with scanning electron microscopy and serial sections of the samples with light microscopy., Key Results: Bract primordia arise first, followed by the stamen primordia in staminate flowers. Both bracts and stamens initiate unidirectionally, first on the abaxial side of the floral apex and later on the adaxial side, most likely due to the contact pressure imposed by the leaf primordium at the superior node. In pistillate flowers, bract primordia on the abaxial side were also initiated first. The configuration of buds at one node showed six patterns and each pattern included at least one vegetative bud, and flower buds were always accompanied by vegetative buds at the same node., Conclusions: The initiation pattern of organs in the outer whorls of C. demersum flowers is distorted by mechanical pressure, resulting in the phyllotactic variation of staminate flowers. Vegetative buds are the main axillary buds with floral buds as accessory buds, which suggests that the shoot of C. demersum has been modified from a decussate phyllotaxis., (© 2015 Botanical Society of America.)

Protoxylem plays an important role in the hydraulic function of vascular systems of both herbaceous and woody plants, but relatively little is known about the processes underlying the maintenance of protoxylem function in long-lived tissues. In this study, embolism repair was investigated in relation to xylem structure in two cushion plant species, Azorella macquariensis and Colobanthus muscoides, in which vascular water transport depends on protoxylem. Their protoxylem vessels consisted of a primary wall with helical thickenings that effectively formed a pit channel, with the primary wall being the pit channel membrane. Stem protoxylem was organized such that the pit channel membranes connected vessels with paratracheal parenchyma or other protoxylem vessels and were not exposed directly to air spaces. Embolism was experimentally induced in excised vascular tissue and detached shoots by exposing them briefly to air. When water was resupplied, embolized vessels refilled within tens of seconds (excised tissue) to a few minutes (detached shoots) with water sourced from either adjacent parenchyma or water-filled vessels. Refilling occurred in two phases: (1) water refilled xylem pit channels, simplifying bubble shape to a rod with two menisci; and (2) the bubble contracted as the resorption front advanced, dissolving air along the way. Physical properties of the protoxylem vessels (namely pit channel membrane porosity, hydrophilic walls, vessel dimensions, and helical thickenings) promoted rapid refilling of embolized conduits independent of root pressure. These results have implications for the maintenance of vascular function in both herbaceous and woody species, because protoxylem plays a major role in the hydraulic systems of leaves, elongating stems, and roots., (© 2015 American Society of Plant Biologists. All Rights Reserved.)

This study focused on determining the phytotoxic mechanism of CeO2 nanoparticles (NPs): destroying chloroplasts and vascular bundles and altering absorption of nutrients on conventional and Bt-transgenic cottons. Experiments were designed with three concentrations of CeO2 NPs including: 0, 100 and 500 mg·L(-1), and each treatment was three replications. Results indicate that absorbed CeO2 nanoparticles significantly reduced the Zn, Mg, Fe, and P levels in xylem sap compared with the control group and decreased indole-3-acetic acid (IAA) and abscisic acid (ABA) concentrations in the roots of conventional cotton. Transmission electron microscopy (TEM) images revealed that CeO2 NPs were absorbed into the roots and subsequently transported to the stems and leaves of both conventional and Bt-transgenic cotton plants via xylem sap. In addition, the majority of aggregated CeO2 NPs were attached to the external surface of chloroplasts, which were swollen and ruptured, especially in Bt-transgenic cotton. The vascular bundles were destroyed by CeO2 nanoparticles, and more damage was observed in transgenic cotton than conventional cotton.

Premise of the Study: Podostemaceae is a unique family of aquatic angiosperms that grow in swift-running water on rock surfaces in the tropics. Their plant bodies show a remarkable adaptation: the main plant body is mostly creeping or flattened, or in extreme cases foliose, functioning as an adhering and photosynthetic organ. In the subfamily Podostemoideae, the root is foliose, whereas in the subfamily Tristichoideae, the shoot is foliose. An evolutionary scenario for the foliose root has already been proposed, but that for the foliose shoot remains to be addressed., Methods: Shoots of Indodalzellia gracilis and Dalzellia ubonensis (subfamily Tristichoideae) were observed using light microscopy and scanning electron microscopy. Gene expression patterns of orthologs of marker genes for the shoot apical meristem, i.e., SHOOT MERISTEMLESS and WUSCHEL, in D. ubonensis were analyzed., Key Results: When very young, the phyllotaxis is tristichous in both genera: a set of one dorsal and two marginal leaves forms. When the shoot branches, extra-axillary buds of two subsequent marginal leaves form as new (lateral) shoots, and the original shoot stops growing; this growth pattern is called sympodial branching. Due to zonal growth in the common zone just below the original and lateral shoot apices, flattened or foliose shoots result. The expression patterns of DuSTM and DuWUS in the shoot apices of Dalzellia were similar to those published for Terniopsis., Conclusions: The foliose shoots of Indodalzellia and Dalzellia evolved as a result of congenital fusion among several original and lateral branches, each of which grows separately in other Tristichoideae., (© 2015 Botanical Society of America, Inc.)

Main Conclusion: Co-cultures of Pinus pinaster with Bursaphelenchus xylophilus were established as a biotechnological tool to evaluate the effect of nematotoxics addition in a host/parasite culture system. The pinewood nematode (PWN), Bursaphelenchus xylophilus, the causal agent of pine wilt disease (PWD), was detected for the first time in Europe in 1999 spreading throughout the pine forests in Portugal and recently in Spain. Plant in vitro cultures may be a useful experimental system to investigate the plant/nematode relationships in loco, thus avoiding the difficulties of field assays. In this study, Pinus pinaster in vitro cultures were established and compared to in vivo 1 year-old plantlets by analyzing shoot structure and volatiles production. In vitro co-cultures were established with the PWN and the effect of the phytoparasite on in vitro shoot structure, water content and volatiles production was evaluated. In vitro shoots showed similar structure and volatiles production to in vivo maritime pine plantlets. The first macroscopic symptoms of PWD were observed about 4 weeks after in vitro co-culture establishment. Nematode population in the culture medium increased and PWNs were detected in gaps of the callus tissue and in cavities developed from the degradation of cambial cells. In terms of volatiles main components, plantlets, P. pinaster cultures, and P. pinaster with B. xylophilus co-cultures were all β- and α-pinene rich. Co-cultures may be an easy-to-handle biotechnological approach to study this pathology, envisioning the understanding of and finding ways to restrain this highly devastating nematode.

Study of plants with unusual phosphorus (P) physiology may assist development of more P-efficient crops. Ptilotus polystachyus grows well at high P supply, when shoot P concentrations ([P]) may exceed 40 mg P g(-1) dry matter (DM). We explored the P physiology of P. polystachyus seedlings grown in nutrient solution with 0-5 mM P. In addition, young leaves and roots of soil-grown plants were used for cryo-scanning electron microscopy and X-ray microanalysis. No P-toxicity symptoms were observed, even at 5 mM P in solution. Shoot DM was similar at 0.1 and 1.0 mM P in solution, but was ∼14% lower at 2 and 5 mM P. At 1 mM P, [P] was 36, 18, 14 and 11 mg P g(-1) DM in mature leaves, young leaves, stems and roots, respectively. Leaf potassium, calcium and magnesium concentrations increased with increasing P supply. Leaf epidermal and palisade mesophyll cells had similar [P]. The root epidermis and most cortical cells had senesced, even in young roots. We conclude that preferential accumulation of P in mature leaves, accumulation of balancing cations and uniform distribution of P across leaf cell types allow P. polystachyus to tolerate very high leaf [P]., (© 2014 John Wiley & Sons Ltd.)

In seed plants, new axes of growth are established by the formation of meristems, groups of pluripotent cells that maintain themselves and initiate the formation of lateral organs. After embryonic development, secondary shoot meristems form in the boundary zones between the shoot apical meristem and leaf primordia, the leaf axils. In addition, many plant species develop ectopic meristems at different positions of the plant body. In the compound tomato leaf, ectopic meristems can initiate at the base of leaflets, which are delimited by two distinct boundary zones, referred to as the proximal (PLB) and distal (DLB) leaflet boundaries. We demonstrate that the two leaflet boundaries differ from each other and that ectopic meristem formation is strictly limited to the DLB. Our data suggest that the DLB harbours a group of pluripotent cells that seems to be the launching pad for meristem formation. Initiation of these meristems is dependent on the activities of the transcriptional regulators Goblet (Gob) and Lateral suppressor (Ls), specifically expressed in the DLB. Gob and Ls act in hierarchical order, because Ls transcript accumulation is dependent on Gob activity, but not vice versa. Ectopic meristem formation at the DLB is also observed in other seed plants, like Cardamine pratensis, indicating that it is part of a widespread developmental program. Ectopic meristem formation leads to an increase in the number of buds, enhances the capacity for survival and opens the route to vegetative propagation., (© 2015 The Authors The Plant Journal © 2015 John Wiley & Sons Ltd.)

Microinjections of fluorescent dyes have revealed that the shoot apical meristem (SAM) is dynamically partitioned into symplasmic fields (SFs), implying that plasmodesmata (Pd) are held shut at specific locations in the proliferating cellular matrix. The SFs are integrated into a coherent morphogenetic unit by exchange of morphogens and transcription factors via gating Pd between adjacent SFs, and by ligand-receptor interactions that operate across the extracellular space. We describe a method for the real-time mapping of SF in the SAM by iontophoresis and membrane potential measurements.

Turions, which are modified shoot apices, are vegetative, dormant overwintering organs produced by perennial aquatic plants. In this study, the turion cytochemistry and ultrastructure of Aldrovanda vesiculosa, Utricularia vulgaris and U. stygia were compared with particular emphasis placed on storage substances. These three aquatic, rootless carnivorous plant species were studied at the end of their winter dormancy. At this stage, the turions of all species had starch as their main storage material. In contrast with A. vesiculosa, Utricularia turions were rich in protein storage vacuoles, and proteins were also accumulated as crystalline inclusions in the nuclei. All examined species accumulated lipid droplets in cells of epidermal glands.

A new symptomatology was observed in celery (Apium graveolens) in Villena, Spain in 2008. Symptomatology included an abnormal amount of shoots per plant and curled stems. These vegetative disorders were associated with 'Candidatus Liberibacter solanacearum' and not with phytoplasmas. Samples from plant sap were immobilized on membranes based on the spot procedure and tested using a newly developed real-time polymerase chain reaction assay to detect 'Ca. L. solanacearum'. Then, a test kit was developed and validated by intralaboratory assays with an accuracy of 100%. Bacterial-like cells with typical morphology of 'Ca. Liberibacter' were observed using electron microscopy in celery plant tissues. A fifth haplotype of 'Ca. L. solanacearum', named E, was identified in celery and in carrot after analyzing partial sequences of 16S and 50S ribosomal RNA genes. From our results, celery (family Apiaceae) can be listed as a new natural host of this emerging bacterium.

Graphene-related research has intensified rapidly in a wide range of disciplines, but few studies have examined ecosystem risks, particularly phytotoxicity. This study revealed that graphene significantly inhibits the number of wheat roots and the biosynthesis of chlorophyll, and altered the morphology of shoots. Humic acid (HA), a ubiquitous form of natural organic matter, significantly (P < 0.05) relieved this phytotoxicity and recovered the sharp morphology of shoot tips. Both graphene and graphene-HA were transferred from wheat roots to shoots and were found in the cytoplasms and chloroplasts. HA increased the disordered structure and surface negative charges, and reduced the aggregation of graphene. HA enhanced the storage of graphene in vacuoles, potentially indicating an effective detoxification path. The content of cadaverine, alkane, glyconic acid, and aconitic acid was up-regulated by graphene, greatly contributing to the observed phytotoxicity. Conversely, inositol, phenylalanine, phthalic acid, and octadecanoic acid were up-regulated by graphene-HA. The metabolic pathway analysis revealed that the direction of metabolic fluxes governed nanotoxicity. This work presents the innovative concept that HA acts as a natural antidote of graphene by regulating its translocation and metabolic fluxes in vivo. This knowledge is critical for avoiding the overestimation of nanomaterial risks and can be used to control nanomaterial contamination.

Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates the cellular process of cryopreserved protocorm-like bodies (PLBs) was different comparative to non-cryopreserved PLBs. The cellular process was not only modified by the freezing and thawing effect but also due to the dehydration process itself during the cryopreservation procedure. Histological observation in Dendrobium Bobby Messina in encapsulation-dehydration method indicated that the degree of plasmolysis causes more cellular changes to the cryopreserved PLBs comparative to non-cryopreserved and stock culture PLBs. These results revealed higher amount of homogenous cell population and denser cytoplasm in cryopreserved PLBs. Histological analysis also revealed more voluminous nucleus in cryopreserved PLBs comparative to non-cryopreserved PLBs and PLBs stock culture. In contrast, scanning electron microscope analysis showed severe damages in cryopreserved PLBs and non-cryopreserved PLBs comparative to the PLBs stock culture which in return could be the possible reason of no regrowth in encapsulation-dehydration method. Damages incurred were on top part, side part, and at the stomata of the PLBs. Histological observation and scanning electron microscopy analyses in Dendrobium Bobby Messina indicates that the degree of plasmolysis causes changes in the cellular process of PLBs from cryopreserved PLBs was different comparative to non-cryopreserved PLBs.

Many studies demonstrate the potential application of arbuscular mycorrhizal fungi (AMF) for remediation purposes, but little is known on AMF potential to enhance plant tolerance to arsenic (As) and the mechanisms involved in this process. We carried anatomical and ultrastructural studies to examine this symbiotic association and the characteristics of shoots and roots of Leucaena leucocephala in As-amended soils (35 and 75 mg As dm(-3)). The experiment used 3 AMF isolates from uncontaminated soils: Acaulospora morrowiae, Glomus clarum, and Gigaspora albida; a mixed inoculum derived from combining these 3 isolates (named Mix AMF); and, 3 AMF isolates from As-contaminated areas: A. morrowiae, G. clarum and Paraglomus occultum. Phytotoxicity symptoms due to arsenic contamination appeared during plant growth, especially in treatments without AMF application. Inoculation with G. clarum and the mixture of species (A. morrowiae, G. albida, and G. clarum) resulted in better growth of L. leucocephala in soils with high As concentrations, as well as significant As removal from the soil, showing a potential for using AMF in phytoextraction. Light microscopy (LS), transmission (TEM) and scanning electron microscopies (SEM) studies showed the colonization of the AMF in plant tissues and damage in all treatments, with ultrastructural changes being observed in leaves and roots of L. leucocephala, especially with the addition of 75 mg dm(-3) of As., (Copyright © 2012 Elsevier B.V. All rights reserved.)

In preparation for cryopreservation, Eucalyptus grandis in vitro axillary buds were dried over silica gel. Pretreatment of the buds with 5 mg per L ABA resulted in partial resistance to water loss (0.76 to 0.45 g per g fresh mass basis) as compared with untreated buds (0.76 to 0.33 g per g) and was associated with the retention of viability (70 vs. 55%). The loss of viability of the dried buds was protracted over several days. Ultrastructural examination and vital staining demonstrated cellular and tissue responses to drying. The meristem appeared to withstand drying and 72 h of rehydration whilst the leaf primordia were destroyed immediately after drying. High reactive oxygen species (ROS) activity was associated with bud excision and drying. Caspase-3-like protease activity was detected after rehydration, thereby providing evidence that the dried buds, that had ultimately died, had undergone programmed cell death. ROS production is considered to be the trigger for programmed cell death.

Leaves of flowering plants are produced from the shoot apical meristem at regular intervals and they grow according to a developmental program that is determined by both genetic and environmental factors. Detailed frameworks for multiscale dynamic analyses of leaf growth have been developed in order to identify and interpret phenotypic differences caused by either genetic or environmental variations. They revealed that leaf growth dynamics are non-linearly and nonhomogeneously distributed over the lamina, in the leaf tissues and cells. The analysis of the variability in leaf growth, and its underlying processes, has recently gained momentum with the development of automated phenotyping platforms that use various technologies to record growth at different scales and at high throughput. These modern tools are likely to accelerate the characterization of gene function and the processes that underlie the control of shoot development. Combined with powerful statistical analyses, trends have emerged that may have been overlooked in low throughput analyses. However, in many examples, the increase in throughput allowed by automated platforms has led to a decrease in the spatial and/or temporal resolution of growth analyses. Concrete examples presented here indicate that simplification of the dynamic leaf system, without consideration of its spatial and temporal context, can lead to important misinterpretations of the growth phenotype., (Copyright © 2013 Wiley Periodicals, Inc.)

Glutathione reductases (GRs) are important components of the antioxidant machinery that plants use to respond against abiotic stresses. In rice, one cytosolic and two chloroplastic GR isoforms have been identified. In this work, we describe the cloning and characterization of the full-length cDNA encoding OsGR3, a chloroplast-localized GR that up to now was considered as a non-functional enzyme because of assumed lack of N-terminal conserved domains. The expression of OsGR3 in E. coli validated that it can be translated as a protein with GR activity. OsGR3 shows 76 and 53 % identity with OsGR1 (chloroplastic) and OsGR2 (cytosolic), respectively. Phylogenetic analysis revealed 2 chloroplastic GRs in Poaceae species, including rice, sorghum and brachypodium, but only one chloroplastic GR in dicots. A plastid transit peptide is located at the N terminus of OsGR3, and genetic transformation of rice with a GR3-GFP fusion construct further confirmed its localization in chloroplasts. Furthermore, OsGR1 and OsGR3 are also targeted to mitochondria, which suggest a combined antioxidant mechanism in both chloroplasts and mitochondria. However, both isoforms showed a distinct response to salinity: the expression of OsGR3 but not OsGR1 was induced by salt stress. In addition, the transcript level of OsGR3 was greatly increased with salicylic acid treatment but was not significantly affected by methyl jasmonate, dehydration or heat shock stress. Our results provide new clues about the possible roles of functional OsGR3 in salt stress and biotic stress tolerance.

Premise: Ginkgo, centrally placed in seed plant phylogeny, is considered important in many phylogenetic and evolutionary studies. Shoot dimorphism of Ginkgo has been long noted, but no work has yet been done to evaluate the relationships between overall branch architecture and wood ring characters, shoot growth, and environmental conditions. •, Methods: Branches, sampled from similar canopy heights, were mapped with the age of each long shoot segment determined by counting annual leaf-scar series on its short shoots. Transverse sections were made for each long shoot segment and an adjacent short shoot; wood ring thickness, number of rings, and number of tracheids/ring were determined. Using branch maps, we identified wood rings for each long shoot segment to year and developmental context of each year (distal short shoot growth only vs. at least one distal long shoot). Climate data were also analyzed in conjunction with developmental context. •, Key Results: Significantly thicker wood rings occur in years with distal long shoot development. The likelihood that a branch produced long shoots in a given year was lower with higher maximum annual temperature. Annual maximum temperature was negatively correlated with ring thickness in microsporangiate trees only. Annual minimum temperatures were correlated differently with ring thickness of megasporangiate and microsporangiate trees, depending on the developmental context. There were no significant effects associated with precipitation. •, Conclusions: Overall, developmental context alone predicts wood ring thickness about as well as models that include temperature. This suggests that although climatic factors may be strongly correlated with wood ring data among many gymnosperm taxa, at least for Ginkgo, correlations with climate data are primarily due to changes in proportions of shoot developmental types (LS vs. SS) across branches.

Premise of the Study: The family Magnoliaceae s.l. is a basal angiosperm family with two subfamilies-Magnolioideae and Liriodendroideae, which differ by the types and structure of their fruits and seeds. The late Albian genus Archaeanthus shares many features of its reproductive organs with Magnoliaceae s.l., but its pericarp anatomy was never studied in detail. A broad-scale carpological investigation of Archaeanthus and Magnoliaceae s.l. was undertaken to reveal the nature of the similarities in fruit structure and to reconstruct Archaeanthus pericarp anatomy. These data are important to determine the early stages of fruit morphogenesis and thus to clarify relationships of Archaeanthus to the taxa of Magnoliaceae s.l., Methods: The pericarp anatomy was studied with light microscopy, SEM, and polarizing microscopy., Key Results: The dehiscent, polyspermous follicles shed from the receptacle of Archaeanthus share similarities with dehiscent follicles of Magnoliaceae s.s. and shedding nutlets of Liriodendron. The seeds of Archaeanthus are dorsiventrally flattened, ovoid, and encircled with a single circular wing. The pericarps of all the taxa studied are differentiated into exocarp (epidermis), multilayered mesocarp, and endocarp (fiber-like sclereids). The mesocarp consists of parenchyma with scattered secretory cells and sclereid clusters (Magnoliaceae s.s., Archaeanthus) or composed by sclerenchyma (Liriodendron)., Conclusions: The specializations of dehiscent multifollicles of unknown Cretaceous ancestors for different modes of seed and fruitlet dispersal formed the basis for the differentiation of two evolutionary lines with their divergence occurring more than 100 million years ago: Magnoliaceae s.s. and the Archaeanthus-Liriodendroidea-Liriodendron line (Liriodendraceae s.l.) within the order Magnoliales.

Image acquisition is an important step in the study of cytoskeleton organization. As visual interpretations and manual measurements of digital images are prone to errors and require a great amount of time, a freely available software package named MicroFilament Analyzer (MFA) was developed. The goal was to provide a tool that facilitates high-throughput analysis to determine the orientation of filamentous structures on digital images in a more standardized, objective and repeatable way. Here, the rationale and applicability of the program is demonstrated by analyzing the microtubule patterns in epidermal cells of control and gravi-stimulated Arabidopsis thaliana roots. Differential expansion of cells on either side of the root results in downward bending of the root tip. As cell expansion depends on the properties of the cell wall, this may imply a differential orientation of cellulose microfibrils. As cellulose deposition is orchestrated by cortical microtubules, the microtubule patterns were analyzed. The MFA program detects the filamentous structures on the image and identifies the main orientation(s) within individual cells. This revealed four distinguishable microtubule patterns in root epidermal cells. The analysis indicated that gravitropic stimulation and developmental age are both significant factors that determine microtubule orientation. Moreover, the data show that an altered microtubule pattern does not precede differential expansion. Other possible applications are also illustrated, including field emission scanning electron micrographs of cellulose microfibrils in plant cell walls and images of fluorescent actin., (© 2013 The Authors The Plant Journal © 2013 John Wiley & Sons Ltd.)

To study if etiolation symptoms exist in plants grown under natural illumination conditions, under-soil epicotyl segments of light-grown pea (Pisum sativum) plants were examined and compared to those of hydroponically dark-grown plants. Light-, fluorescence- and electron microscopy, 77 K fluorescence spectroscopy, pigment extraction and pigment content determination methods were used. Etioplasts with prolamellar bodies and/or prothylakoids, protochlorophyll (Pchl) and protochlorophyllide (Pchlide) forms (including the flash-photoactive 655 nm emitting form) were found in the (pro)chlorenchyma of epicotyl segments under 3 cm soil depth; their spectral properties were similar to those of hydroponically grown seedlings. However, differences were found in etioplast sizes and Pchlide:Pchl molar ratios, which indicate differences in the developmental rates of the under-soil and of hydroponically developed cells. Tissue regions closer to the soil surface showed gradual accumulation of chlorophyll, and in parallel, decrease of Pchl and Pchlide. These results proved that etioplasts and Pchlide exist in soil-covered parts of seedlings even if they have a 3-4-cm long photosynthetically active shoot above the soil surface. This underlines that etiolation symptoms do develop under natural growing conditions, so they are not merely artificial, laboratory phenomena. Consequently, dark-grown laboratory plants are good models to study the early stages of etioplast differentiation and the Pchlide-chlorophyllide phototransformation., (Copyright © Physiologia Plantarum 2012.)

Differential centrifugation and synchrotron radiation X-ray fluorescence spectroscopy (SRXRF) microprobe were used to study the distribution of the elements in tissue cross sections of pakchoi ( Brassica chinensis L.) under stress of elevated Pb and Cr. Subcellular fractionation of the different tissues grown in a nutrient solution containing 200 mg L(-1) Pb or 5 mg L(-1) Cr showed that 86.7 and 76.3% of the Pb that accumulated in the roots and shoots, respectively, was contained in the cell wall and vacuoles in those areas. Whereas 75.0% of the Cr that accumulated in the root was contained in the cell wall, 63.1% of the Cr that accumulated in the shoot was found in the vacuoles and cell wall. SRXRF analysis revealed that, when pakchoi seedlings were placed under excess Pb stress, the Pb, Ca, Cu, and Zn were concentrated in the cortex and vascular bundle of the root and mixed Fe-Mn plaques were seen on the surface of the pakchoi root. In the Cr treatment group, Cr, Ca, Mn, and Zn were mainly located in the cortex of the root, whereas in the stem, only Ca, Cu, and Zn were detected at higher levels in the cortex area. Thus, this study provides evidence that, in response to Pb and Cr stress, pakchoi uses cell walls and vacuoles to reduce the transport of these heavy metals through the plant, as well as restrict transport from the root to the stem.

Drought stress responses and sensitivity of dioecious plants, such as Populus cathayana Rehd., are determined by different mechanisms in each sex. In general, males tend to be more resistant while females are more sensitive. Here, we used reciprocal grafting between males and females to determine the relative importance of roots and shoots when plants are exposed to drought stress. Total dry matter accumulation (DMA), photosynthetic capacity, long-term water-use efficiency (Δ), water potential and ultrastructure of mesophyll cells were evaluated to determine the different roles of root and shoot in sex-related drought responses. Plants with male roots were found to be more resistant and less sensitive to water stress than those with female roots under drought conditions. On the contrary, plants with female shoots grew better than those with male shoots under well-watered conditions. These results indicated that the sensitivity of males and females to water stress is primarily influenced by root processes, while under well-watered conditions sexual differences in growth are primarily driven by shoot processes. Furthermore, grafting female shoot scion onto male rootstock was proved to be an effective mean to improve resistance to water stress in P. cathayana females., (© 2012 Blackwell Publishing Ltd.)