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The apoplastic pH (pH Apo ) in plants is susceptible to environmental stimuli. However, the biological implications of pH Apo variation have remained largely unknown. The universal stress phytohormone abscisic acid (ABA) as well as the major environmental stimuli drought and salinity were selected as representative cases to investigate how changes in pH Apo relate to plant behaviors in Arabidopsis. Variations in pH Apo negatively regulated the cotyledon greening inhibition to the universal stress hormone ABA or environmental stimuli through the action of extracellular hydrogen peroxide (eH 2 O 2 ). Further studies revealed that an increase in pH Apo diminishes the chemical reactivity of eH 2 O 2 , effectively functioning as an 'off' switch for its action in oxidizing thiols of plasma membrane proteins. Consequently, this suppresses the eH 2 O 2 -mediated cotyledon greening inhibition to environmental stimuli and ABA, alongside inhibiting the eH 2 O 2 -mediated intracellular Ca 2+ signaling. Conversely, a decrease in pH Apo serves as an 'on' switch for the action of eH 2 O 2 . In summary, the pH Apo is a crucial messenger and chemical switch for eH 2 O 2 in signal transduction, notwithstanding the apparent simplicity of the underlying mechanism. Our findings provide a novel fundamental biological insight into the significance of pH., (© 2025 The Author(s). New Phytologist © 2025 New Phytologist Foundation.) more...

Low-temperature (LT) stress seriously affects the distribution, seedling survival, and grain yield of maize. At the seedling emergence stage, maize's coleoptile is one of the most sensitive organs in sensing LT signaling and, in general, it can envelop young leaves to protect them from LT damage. In addition, brassinolides (BRs) have been shown to enhance LT tolerance from various species, but the effects of BRs on coleoptiles in maize seedlings under LT stress are unclear. Therefore, in this study, the pre-cultured coleoptiles of Zheng58 seedlings were treated with or without 2.0 μM 24-epibrassinolide (EBR) at 25 °C and 10 °C environments for five days to analyze their physiological and transcriptomic changes. Physiological analysis showed that a 10°C LT stress increased the content of glucose (0.43 mg g -1 FW), sucrose (0.45 mg g -1 FW), and starch (0.76 mg g -1 FW) of Zheng58 coleoptiles compared to a 25°C environment. After the coleoptiles were exposed to a 2.0 μM EBR application under 10°C temperature for five days, the contents of these three sugars continued to increase, and reached 2.68 mg g -1 FW, 4.64 mg g -1 FW, and 9.27 mg g -1 FW, respectively, indicating that sugar signaling and metabolism played key roles in regulating LT tolerance in the coleoptiles of maize seedlings. Meanwhile, a transcriptome analysis showed that 84 and 15 differentially expressed genes (DEGs) were enriched in the sucrose and starch metabolism and photosynthesis pathways, respectively, and multiple DEGs involved in these pathways were significantly up-regulated under LT stress and EBR stimulation. Further analysis speculated that the four DEGs responsible for sucrose-phosphate synthetase (SPS, i.e., Zm00001d048979 , probable sucrose-phosphate synthase 5 and Zm00001d012036 , sucrose-phosphate synthase 1), sucrose synthase (SUS, Zm00001d029091 , sucrose synthase 2 and Zm00001d029087 , sucrose synthase 4) were crucial nodes that could potentially link photosynthesis and other unknown pathways to form the complex interaction networks of maize LT tolerance. In conclusion, our findings provide new insights into the molecular mechanisms of exogenous EBR in enhancing LT tolerance of maize seedlings and identified potential candidate genes to be used for LT tolerance breeding in maize. more...

Capsicum annuum L. is extensively cultivated in subtropical and temperate regions globally, respectively, when grown in a medium with 8 holding significant economic importance. Despite the availability of genome sequences and editing tools, gene editing in peppers is limited by the lack of a stable regeneration and transformation method. This study assessed regeneration and transformation protocols in seven chili pepper varieties, including CM334, Zunla-1, Zhongjiao6 (ZJ6), 0818, 0819, 297, and 348, in order to enhance genetic improvement efforts. Several explants, media compositions, and hormonal combinations were systematically evaluated to optimize the in vitro regeneration process across different chili pepper varieties. The optimal concentrations for shoot formation, shoot elongation, and rooting in regeneration experiments were determined as 5 mg/L of 6-Benzylaminopurine (BAP) with 5 mg/L of silver nitrate (AgNO 3 ), 0.5 mg/L of Gibberellic acid (GA 3 ), and 1 mg/L of Indole-3-butyric acid (IBA), respectively. The highest regeneration rate of 41% was observed from CM334 cotyledon explants. Transformation optimization established 300 mg/L of cefotaxime for bacterial control, with a 72-h co-cultivation period at OD 600 = 0.1. This study optimizes the protocols for chili pepper regeneration and transformation, thereby contributing to genetic improvement efforts. more...

This study explores the impact of juglone on cucumber (Cucumis sativus cv. Beith Alpha), scrutinizing its effects on seed germination, growth, and the polyphenol oxidase (PPO) enzyme's activity and gene expression. Employing concentrations ranging from 0.01 to 0.5 mM, we found juglone's effects to be concentration-dependent. At lower concentrations (0.01 and 0.1 mM), juglone promoted root and shoot growth along with germination, whereas higher concentrations (0.25 and 0.5 mM) exerted inhibitory effects, delineating a threshold for its allelopathic influence. Notably, PPO activity surged, especially at 0.5 mM in roots, hinting at oxidative stress involvement. Real-time PCR unveiled that juglone modulates PPO gene expression in cotyledons, peaking at 0.1 mM and diminishing at elevated levels. Correlation analyses elucidated a positive link between juglone-induced root growth and cotyledon PPO gene expression but a negative correlation with heightened root enzyme activity. Additionally, germination percentage inversely correlated with root PPO activity, while PPO activities positively associated with dopa and catechol substrates in both roots and cotyledons. Molecular docking studies revealed juglone's selective interactions with PPO's B chain, suggesting regulatory impacts. Protein interaction assessments highlighted juglone's influence on amino acid metabolism, and molecular dynamics indicated juglone's stronger, more stable binding to PPO, inferring potential alterations in enzyme function and stability. Conclusively, our findings elucidate juglone's dose-dependent physiological and biochemical shifts in cucumber plants, offering insights into its role in plant growth, stress response, and metabolic modulation., (© 2024 The Author(s). Physiologia Plantarum published by John Wiley & Sons Ltd on behalf of Scandinavian Plant Physiology Society.) more...

Brassinosteroids (BRs) are phytohormones that regulate stomatal development. In this study, we report that BR represses stomatal development in etiolated Arabidopsis (Arabidopsis thaliana) cotyledons via transcription factors BRASSINAZOLE RESISTANT 1 (BZR1) and bri1-EMS SUPPRESSOR1 (BES1), which directly target MITOGEN-ACTIVATED PROTEIN KINASE KINASE 9 (MKK9) and FAMA, 2 important genes for stomatal development. BZR1/BES1 bind MKK9 and FAMA promoters in vitro and in vivo, and mutation of the BZR1/BES1 binding motif in MKK9/FAMA promoters abolishes their transcription regulation by BZR1/BES1 in plants. Expression of a constitutively active MKK9 (MKK9DD) suppressed overproduction of stomata induced by BR deficiency, while expression of a constitutively inactive MKK9 (MKK9KR) induced high-density stomata in bzr1-1D. In addition, bzr-h, a sextuple mutant of the BZR1 family of proteins, produced overabundant stomata, and the dominant bzr1-1D and bes1-D mutants effectively suppressed the stomata-overproducing phenotype of brassinosteroid insensitive 1-116 (bri1-116) and brassinosteroid insensitive 2-1 (bin2-1). In conclusion, our results revealed important roles of BZR1/BES1 in stomatal development, and their transcriptional regulation of MKK9 and FAMA expression may contribute to BR-regulated stomatal development in etiolated Arabidopsis cotyledons., Competing Interests: Conflict of interest statement. None declared., (© The Author(s) 2024. Published by Oxford University Press on behalf of American Society of Plant Biologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.) more...

Somatic embryogenesis is an effective tool for the production of forest tree seedlings with desirable characteristics; however, the low initiation frequency and productivity of high-quality mature somatic embryos are still limiting factors for Larix kaempferi (Japanese larch). Here, we analyzed the expression pattern of L . kaempferi   cyclin-dependent kinase B 1;2 ( LaCDKB1;2 ) during somatic embryogenesis in L. kaempferi and its relationship with the cell proliferation rate. We also analyzed the effect of LaCDKB1;2 over-expression on somatic embryo quality. The results revealed a positive correlation between LaCDKB1;2 expression and the cell proliferation rate during the proliferation stage. After LaCDKB1;2 over-expression, the proliferation rate of cultures increased, and the number of somatic embryos in transgenic cultures was 2.69 times that in non-transformed cultures. Notably, the number of normal cotyledonary embryos in transgenic cultures was 3 times that in non-transformed cultures, indicating that LaCDKB1;2 not only increases the proliferation of cultures and the number of somatic embryos but also improves the quality of somatic embryos. These results provide insight into the regulatory mechanisms of somatic embryogenesis as well as new Larix breeding material. more...

In plants, the effective mobilization of seed nutrient reserves is crucial during germination and for seedling establishment. The Arabidopsis H+-PPase-loss-of-function fugu5 mutants exhibit a reduced number of cells in the cotyledons. This leads to enhanced post-mitotic cell expansion, also known as compensated cell enlargement (CCE). While decreased cell numbers have been ascribed to reduced gluconeogenesis from triacylglycerol, the molecular mechanisms underlying CCE remain ill-known. Given the role of indole 3-butyric acid (IBA) in cotyledon development, and because CCE in fugu5 is specifically and completely cancelled by ech2, which shows defective IBA-to-indoleacetic acid (IAA) conversion, IBA has emerged as a potential regulator of CCE. Here, to further illuminate the regulatory role of IBA in CCE, we used a series of high-order mutants that harbored a specific defect in IBA-to-IAA conversion, IBA efflux, IAA signaling, or vacuolar type H+-ATPase (V-ATPase) activity and analyzed the genetic interaction with fugu5-1. We found that while CCE in fugu5 was promoted by IBA, defects in IBA-to-IAA conversion, IAA response, or the V-ATPase activity alone cancelled CCE. Consistently, endogenous IAA in fugu5 reached a level 2.2-fold higher than the WT in 1-week-old seedlings. Finally, the above findings were validated in icl-2, mls-2, pck1-2 and ibr10 mutants, in which CCE was triggered by low sugar contents. This provides a scenario in which following seed germination, the low-sugar-state triggers IAA synthesis, leading to CCE through the activation of the V-ATPase. These findings illustrate how fine-tuning cell and organ size regulation depend on interplays between metabolism and IAA levels in plants., Competing Interests: The authors have declared that no competing interests exist. more...

The fungal toxin fusicoccin (FC) induces rapid cell elongation, proton extrusion and plasma membrane hyperpolarization in maize coleoptile cells. Here, these three parameters were simultaneously measured using non-abraded and non-peeled segments with the incubation medium having access to their lumen. The dose-response curve for the FC-induced growth was sigmoidal shaped with the maximum at 10 -6 M over 10 h. The amplitudes of the rapid growth and proton extrusion were significantly higher for FC than those for indole-3-acetic acid (IAA). The differences between the membrane potential changes that were observed in the presence of FC and IAA relate to the permanent membrane hyperpolarization for FC and transient hyperpolarization for IAA. It was also found that the lag times of the rapid growth, proton extrusion and membrane hyperpolarization were shorter for FC compared to IAA. At 30 °C, the biphasic kinetics of the IAA-induced growth rate could be changed into a monophasic (parabolic) one, which is characteristic for FC-induced rapid growth. It has been suggested that the rates of the initial phase of the FC- and IAA-induced growth involve two common mechanisms that consist of the proton pumps and potassium channels whose contribution to the action of both effectors on the rapid growth is different. more...

Seedlings grown in darkness exhibit distinct morphologies comparing with light-grown seedlings. Elongated hypocotyls, closed yellow cotyledons, and the formation of apical hooks are typical characteristics for etiolated seedlings, which are collectively named skotomorphogenesis. Various plant hormones and environmental factors are essential for maintaining skotomorphogenesis. Due to the diverse morphological outcomes in etiolated seedlings grown under different treatments, studies on skotomorphogenesis are of particular importance to reveal the molecular mechanisms underlying plant response to environmental cues. Here, we detailed experimental procedures to facilitate researchers who are investigating etiolation growth-related studies. more...

After germination, plants determine their morphogenesis, such as hypocotyl elongation and cotyledon opening, by responding to various wavelengths of light (photomorphogenesis). Cryptochrome is a blue-light photoreceptor that controls de-etiolation, stomatal opening and closing, flowering time, and shade avoidance. Successful incorporation of these phenotypes as indicators into a chemical screening system results in faster selection of candidate compounds. Here, we describe phenotypic screening for the blue-light response of Arabidopsis thaliana seedling and the resulting process that clarifies that the compound obtained in the screening is an inhibitor of cryptochromes. more...

Nicotinamide adenine dinucleotide (NAD) plays a central role in redox metabolism in all domains of life. Additional roles in regulating posttranslational protein modifications and cell signaling implicate NAD as a potential integrator of central metabolism and programs regulating stress responses and development. Here we found that NAD negatively impacts stomatal development in cotyledons of Arabidopsis thaliana. Plants with reduced capacity for NAD + transport from the cytosol into the mitochondria or the peroxisomes exhibited reduced numbers of stomatal lineage cells and reduced stomatal density. Cotyledons of plants with reduced NAD + breakdown capacity and NAD + -treated cotyledons also presented reduced stomatal number. Expression of stomatal lineage-related genes was repressed in plants with reduced expression of NAD + transporters as well as in plants treated with NAD + . Impaired NAD + transport was further associated with an induction of abscisic acid (ABA)-responsive genes. Inhibition of ABA synthesis rescued the stomatal phenotype in mutants deficient in intracellular NAD + transport, whereas exogenous NAD + feeding of aba-2 and ost1 seedlings, impaired in ABA synthesis and ABA signaling, respectively, did not impact stomatal number, placing NAD upstream of ABA. Additionally, in vivo measurement of ABA dynamics in seedlings of an ABA-specific optogenetic reporter - ABAleon2.1 - treated with NAD + showed increases in ABA content suggesting that NAD + impacts on stomatal development through ABA synthesis and signaling. Our results demonstrate that intracellular NAD + homeostasis as set by synthesis, breakdown and transport is essential for normal stomatal development, and provide a link between central metabolism, hormone signaling and developmental plasticity., (© 2020 Society for Experimental Biology and John Wiley & Sons Ltd.) more...

Histone acetylation plays an important role in plant growth and development. Here, we investigated the effect of sodium butyrate (NaB), a histone deacetylase inhibitor, on adventitious shoot formation from protoplast-derived calli and cotyledon explants of tobacco ( Nicotiana benthamiana ) and tomato ( Solanum lycopersicum ). The frequency of adventitious shoot formation from protoplast-derived calli was higher in shoot induction medium (SIM) containing NaB than in the control. However, the frequency of adventitious shoot formation from cotyledon explants of tobacco under the 0.1 mM NaB treatment was similar to that in the control, but it decreased with increasing NaB concentration. Unlike in tobacco, NaB decreased adventitious shoot formation in tomato explants in a concentration-dependent manner, but it did not have any effect on adventitious shoot formation in calli. NaB inhibited or delayed the expression of D-type cyclin ( CYCD3-1 ) and shoot-regeneration regulatory gene WUSCHEL ( WUS ) in cotyledon explants of tobacco and tomato. However, compared to that in control SIM, the expression of WUS was promoted more rapidly in tobacco calli cultured in NaB-containing SIM, but the expression of CYCD3-1 was inhibited. In conclusion, the effect of NaB on adventitious shoot formation and expression of CYCD3-1 and WUS genes depended on the plant species and whether the effects were tested on explants or protoplast-derived calli. more...

The plant-specific receptor-like cytoplasmic kinases (RLCKs) form a large, poorly characterized family. Members of the RLCK VI_A class of dicots have a unique characteristic: their activity is regulated by Rho-of-plants (ROP) GTPases. The biological function of one of these kinases was investigated using a T-DNA insertion mutant and RNA interference. Loss of RLCK VI_A2 function resulted in restricted cell expansion and seedling growth. Although these phenotypes could be rescued by exogenous gibberellin, the mutant did not exhibit lower levels of active gibberellins nor decreased gibberellin sensitivity. Transcriptome analysis confirmed that gibberellin is not the direct target of the kinase; its absence rather affected the metabolism and signalling of other hormones such as auxin. It is hypothesized that gibberellins and the RLCK VI_A2 kinase act in parallel to regulate cell expansion and plant growth. Gene expression studies also indicated that the kinase might have an overlapping role with the transcription factor circuit (PIF4-BZR1-ARF6) controlling skotomorphogenesis-related hypocotyl/cotyledon elongation. Furthermore, the transcriptomic changes revealed that the loss of RLCK VI_A2 function alters cellular processes that are associated with cell membranes, take place at the cell periphery or in the apoplast, and are related to cellular transport and/or cell wall reorganisation., Competing Interests: The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. more...

Adventitious roots form only at the proximal cut surface (PCS) but not at the distal cut surface (DCS) of mango cotyledon segments. In this study, mango embryos treated with indole-3-butyric acid (IBA) showed significantly increased adventitious root formation, while those treated with 2, 3, 5-triiodobenzoic acid (TIBA) demonstrated complete inhibition of adventitious rooting. Mango embryos treated with auxin influx inhibitors demonstrated lower inhibition of adventitious roots than those treated with TIBA. The endogenous indol-3-acetic acid (IAA) content on the PCS and DCS was similar at 0 h, then increased on both surfaces after 6 h, and IAA content on the PCS were always higher than those on the DCS. We cloned three genes encoding auxin efflux carriers (i.e., MiPIN2-4) and examined their temporal and spatial expression patterns under different treatments. Relative expression of all MiPINs studied was very low at 0 h but significantly increased on both PCS and DCS from 1 d to 10 d, to varying degrees. We overexpressed MiPIN1-4 in Arabidopsis plants and found a significant increase in adventitious root quantity in MiPIN1 and MiPIN3 transgenic lines. Immunofluorescence results showed that MiPIN1 and MiPIN3 are primarily localized in the vascular tissues and the cells adjacent to abaxial surface. In conclusion, we propose that in mango cotyledon segments, wounding stimulates IAA biosynthesis, the transcription levels of PIN genes were significantly increased in different magnitudes on the PCS and DCS, resulting in polar IAA transport from the DCS to PCS via the vascular tissues, thereby triggering adventitious root formation., Competing Interests: Declaration of competing interest The authors declare that they have no conflict of interest., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.) more...

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

Hormesis, which describes the stimulatory effect of low doses of toxic substances on growth, is a well-known phenomenon in the plant and animal kingdoms. However, the mechanisms that are involved in this phenomenon are still poorly understood. We performed preliminary studies on corn coleoptile sections, which showed a positive correlation between the stimulation of growth by Cd or Pb and an increase in the auxin and H 2 O 2 content in the coleoptile sections. Subsequently, we grew corn seedlings in hydroponic culture and tested a wide range of Cd or Pb concentrations in order to determine hormetic growth stimulation. In these seedlings the gas exchange and the chlorophyll a fluorescence, as well as the content of chlorophyll, flavonol, auxin and hydrogen peroxide, were measured. We found that during the hormetic stimulation of growth, the response of the photosynthetic apparatus to Cd and Pb differed significantly. While the application of Cd mostly caused a decrease in various photosynthetic parameters, the application of Pb stimulated some of them. Nevertheless, we discovered that the common features of the hormetic stimulation of shoot growth by heavy metals are an increase in the auxin and flavonol content and the maintenance of hydrogen peroxide at the same level as the control plants. more...

In this works, a simple, efficient and repeatable protocol was developed for in vitro regeneration via callus-mediated organogenesis of Neolamarkia Cadamba using cotyledonary petioles and hypocotyls. Effects of basal medium, plant growth regulators, the types and age of explant on the formation of adventitious buds/shoots were studied. Meanwhile, histological analysis for early ontogenic stages and genetic stability assessment by flow cytometry were investigated. Our investigation demonstrated that, compared with 6-benzyladenine (BA), N 6 -(2-isopentenyl) adenine (2-ip), Thidiazuron (TDZ) was the optimal cytokinin for buds/shoots induction on cotyledon and hypocotyl explants. Douglas-fir and sugar pine medium (DCR) supplemented with 22.7 μM TDZ and 0.27 μM α-naphthalene acetic acid (NAA) was most effective on bud induction, with the highest bud-induction rate and numbers of buds on cotyledon and hypocotyl explants. The available shoot per explant hit 35.2 when the induced callus sub-cultured to a medium without TDZ. It was found that TDZ could promote induction of the callus and the buds, however, continuous exposure beyond 4 weeks of supplemented high concentration (exceed 11.35 μM), TDZ was harmful to the proliferation and growth of buds/shoots. DCR appeared more efficiency than Murashige and Skoog medium (MS), Woody Plant medium (WPM), anther culture of cereal crops medium (N 6 ) on bud induction. Age of cotyledon and hypocotyl explants in 20-day to 25-day was most beneficial to adventitious buds/shoots formation. Histological investigation confirmed that the buds originated from the wounded incisions of cotyledonary petiole and hypocotyl fragments, with callus formation. The regeneration plantlets were successfully acclimatized in greenhouse, yielded above 95% survival rate in field, exhibited normal morphology and growth characteristics. The analysis of flow cytometry on N. cadamba indicated no variation in the ploidy levels between the regenerated plantlets and the donor trees. The developed procedure can be used for mass production, germplasm exchange and transgenic studies to improve the resistance of the species via Agrobacterium-mediated. more...

Seedling establishment consists of the former stage (i.e. skotomorphogenesis) and the latter stage (i.e. photomorphogenesis). Due to specific developmental processes in plants, the two stages may have different sensitivities to antibiotics. Tetracycline (TC), for example, is a major-use antibiotic. Radicle length, the relatively sensitive endpoint in plant skotomorphogenesis, is less sensitive than all of the indices of cotyledon colour and pigments in plant photomorphogenesis to TC stress. In conclusion, we suggest that plant photomorphogenesis may be more sensitive than plant skotomorphogenesis to stresses of antibiotics, but which needs further studies., (Copyright © 2019 Elsevier Ltd. All rights reserved.) more...

Cultivating rice in wet or water direct seeding systems is simple and time and labor efficient. Rice (Oryza sativa) seeds are a unique cereal that can germinate not only when submerged, but also in anoxic conditions. Many complicated hormone signals interact in submerged seed germination. Ethylene is involved in rice coleoptile elongation, but little is known regarding the role of auxin signaling under submergence. This study demonstrated that the coleoptile is shorter and curlier when submerged with 2,3,5-triiodobenzoic acid (TIBA). In transcriptomic analysis, 3448 of the 31,860 genes were upregulated, and 4360 genes were downregulated with submergence and TIBA treatment. The Gene Ontology function classification results demonstrated that upregulated differentially expressed genes (DEGs) were mainly involved in redox, stress, and signal transduction, whereas the down-regulated DEGs were mainly involved in RNA transcription, stress, and development. Furthermore, auxin signaling involved in the carbohydrate metabolism pathway was demonstrated while using transcriptomic analysis and confirmed in a quantitative real-time polymerase chain reaction. In addition, the transcript levels of development-related genes and mitochondria-electron- transport-related genes were regulated by auxin signaling under submergence. Auxin signaling was not only involved in regulating rice coleoptile elongation and development, but also regulated secondary metabolism, carbohydrate metabolism, and mitochondria electron transport under submergence. Our results presented that auxin signaling plays an important role during rice coleoptile elongation upon the submergence condition and improving the advance of research of direct rice seeding system., Competing Interests: The authors declare that they have no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. more...

Background: Efficient organogenesis induction in eggplant (Solanum melongena L.) is required for multiple in vitro culture applications. In this work, we aimed at developing a universal protocol for efficient in vitro regeneration of eggplant mainly based on the use of zeatin riboside (ZR). We evaluated the effect of seven combinations of ZR with indoleacetic acid (IAA) for organogenic regeneration in five genetically diverse S. melongena and one S. insanum L. accessions using two photoperiod conditions. In addition, the effect of six different concentrations of indolebutyric acid (IBA) in order to promote rooting was assessed to facilitate subsequent acclimatization of plants. The ploidy level of regenerated plants was studied., Results: In a first experiment with accessions MEL1 and MEL3, significant (p < 0.05) differences were observed for the four factors evaluated for organogenesis from cotyledon, hypocotyl and leaf explants, with the best results obtained (9 and 11 shoots for MEL1 and MEL3, respectively) using cotyledon tissue, 16 h light / 8 h dark photoperiod conditions, and medium E6 (2 mg/L of ZR and 0 mg/L of IAA). The best combination of conditions was tested in the other four accessions and confirmed its high regeneration efficiency per explant when using both cotyledon and hypocotyl tissues. The best rooting media was R2 (1 mg/L IBA). The analysis of ploidy level revealed that between 25 and 50% of the regenerated plantlets were tetraploid., Conclusions: An efficient protocol for organogenesis of both cultivated and wild accessions of eggplant, based on the use of ZR, is proposed. The universal protocol developed may be useful for fostering in vitro culture applications in eggplant requiring regeneration of plants and, in addition, allows developing tetraploid plants without the need of antimitotic chemicals. more...

Seedling establishment is a critical step in environment colonisation by higher plants that frequently occurs under adverse conditions. Thus, we carried out an integrated analysis of seedling growth, water status, ion accumulation, reserve mobilisation, metabolite partitioning and hydrolase activity during seedling establishment of the native Caatinga species Piptadenia moniliformis (Benth.) Luckow & R.W. Jobson under salinity. Two-day-old seedlings were cultivated in vitro for 4 days in water agar (control) or supplemented with 50 or 100 mm NaCl. Biochemical determinations were performed according to standard spectrophotometric protocols. We found that 100 mm NaCl stimulated starch degradation, amylase activity and soluble sugar accumulation, but limited storage protein hydrolysis in the cotyledons of P. moniliformis seedlings. Although Na + accumulation in the seedling affected K + partitioning between different organs, it was not possible to associate the salt-induced changes in reserve mobilisation with Na + toxicity, or water status, in the cotyledons. Remarkably, we found that starch content increased in the roots of P. moniliformis seedlings under 100 mm NaCl, probably in response to the toxic effects of Na + . The mobilisation of carbon and nitrogen reserves is independently regulated in P. moniliformis seedlings under salt stress. The salt-induced delay in seedling establishment and the resulting changes in the source-sink relationship may lead to storage protein retention in the cotyledons. Possibly, the intensification of starch mobilisation in the cotyledons supported starch accumulation in the root as a potential mechanism to mitigate Na + toxicity., (© 2019 German Society for Plant Sciences and The Royal Botanical Society of the Netherlands.) more...

Chemical communication underlies major adaptive traits in plants and shapes the root microbiome. An increasing number of diffusible and/or volatile organic compounds released by bacteria have been identified, which play phytostimulant or protective functions, including dimethyl-hexa-decylamine (DMHDA), a volatile biosynthesized by Arthrobacter agilis UMCV2 that induces jasmonic acid (JA) signaling in Arabidopsis. Here, he found that the growth repressing effects of both DMHDA and JA are antagonized by kinetin and correlated with an inhibition of cytokinin-related ARR5::GUS and TCS::GFP expression in Arabidopsis primary roots. Moreover, we demonstrate that shoot supplementation of JA triggers JAZ1 expression both locally and systemically and represses cytokinin-dependent promoter activity in roots. A similar effect was observed after cotyledon wounding, in which an increase of JA-inducible LOX2:GUS expression represses root growth, which correlates with the loss of TCS::GFP detection at the very root tip. Our data demonstrate that the bacterial volatile DMHDA crosstalks with cytokinin signaling and reveals the downstream antagonistic interaction between JA and cytokinin in controlling root growth. more...

Naphthoquinones, plants secondary metabolites are known for their antibacterial, antifungal, anti-inflammatory, anti-cancer and anti-parasitic properties. The biological activity of naphthoquinones is connected with their ability to generate reactive oxygen species and to modify biological molecules at their nucleophilic sites. In our research, the effect of naphthazarin (DHNQ) combined with 2-hydroxy-1,4-naphthoquinone (NQ-2-OH) or 1,4-naphthoquinone (1,4-NQ) on the elongation growth, pH changes of the incubation medium, oxidative stress and redox activity of maize coleoptile cells were investigated. This paper describes experiments performed with maize ( Zea mays L.) coleoptile segments, which is a classical model system to study plant cell elongation growth. The data presented clearly demonstrate that lawsone and 1,4-naphthoquinone combined with naphthazarin, at low concentrations (1 and 10 nM), reduced the endogenous and IAA-induced (Indole-3-Acetic Acid) elongation growth of maize coleoptile segments. Those changes in growth correlated with the proton concentration in the incubation medium, which suggests that the changes in the growth of maize coleoptile segments observed in the presence of naphthoquinones are mediated through the activity of PM H⁺-ATPase. The presence of naphthoquinones induced oxidative stress in the maize coleoptile tissue by producing hydrogen peroxide and causing changes in the redox activity. Moreover, the incubation of maize segments with both naphthoquinones combined with naphthazarin resulted in lipid peroxidation and membrane damage. The regulation of PM H⁺-ATPase activity, especially its inhibition, may result from two major types of reaction: first, a direct interaction between an enzyme and naphthoquinone, which leads to the covalent modification of the protein thiols and the generation of thioethers, which have been found to alter the activity of the PM H⁺-ATPases; second, naphthoquinones induce reactive oxygen species (ROS) production, which inhibits PM H⁺-ATPases by increasing cytosolic Ca 2+ . This harmful effect was stronger when naphthazarin and 1,4-naphthoquinone were added together. Taking these results into account, it can be suggested that by combining naphthoquinones in small quantities, an alternative to synthetic pesticides could be developed. more...

Free polyamine (PA) titers in plants may be regulated through reversible conjugate formation and/or through modulation of their synthesis, transport and degradation. PA signaling involves the well-acknowledged signaling molecule, nitric oxide (NO), which functions in diverse biological processes. Present investigations demonstrate the influence of salt stress (120 mM NaCl) and exogenous NO donor (250 µM Diethylenetriamine, DETA) on PA homeostasis of 2 d old, etiolated sunflower ( Helianthus annuus L.) seedling cotyledons as a long-distance signaling response. Significantly enhanced intracellular spermine (Spm) accumulation was observed in seedling cotyledons under salt stress and in response to NO donor, the increase being more pronounced in seedlings treated with NO, evidently as a result of upregulation of the PA biosynthetic enzymes - arginine decarboxylase (ADC) and S -adenosylmethionine decarboxylase (SAMDC) - as revealed by Western blot and confocal imaging (CLSM). Moreover, salt stress induced the activity of polyamine oxidase (PAO), a PA catabolic enzyme, while NO lowered its activity in salt-stressed seedling cotyledons. NO, thus, appears to assist the seedlings in adapting to salt stress by positively regulating PA homeostasis through regulation of PA distribution between free, conjugated and bound forms, increased accumulation of PA biosynthetic enzymes and lowering the rate of PA catabolism. more...

The mechanisms of the toxic effects of Cd and Pb on plant cell growth are still poorly understood. In particular, little is known about their interactive effects, which usually occur in the environment. Moreover, the data that do exist in the literature are controversial. This study describes experiments that were performed with maize (Zea mays) coleoptile segments, which is a classical model system for studies of plant cell elongation growth. Cadmium and lead, which were added at 0.1 mM, reduced the endogenous and IAA-induced elongation growth of maize coleoptile cells. When both metals were added together or in sequence, their effect on IAA-induced growth was more toxic. The medium pH changes, which were measured simultaneously with growth, indicated that while Pb stopped IAA-induced proton extrusion, Cd only partially diminished it. Although Cd was generally more accumulated than Pb in the maize coleoptile segments, when IAA was added together with Pb, it significantly increased the accumulation of the metal. The short-term electrophysiological experiments showed that the addition of Cd caused the depolarisation of the membrane potential (E m ), whereas Pb caused membrane hyperpolarisation. In the long-term electrophysiological experiments, it was found that the Cd-induced E m changes are complex. In conclusion, these results suggest that the effects of Cd and Pb as well as their combination on the elongation growth of maize coleoptile cells and the accumulation of the metals result, among others, from different ionic mechanisms by which each metal change the membrane potential of the cells., (Copyright © 2018 Elsevier Inc. All rights reserved.) more...

Stilbene synthase genes make a contribution to improving the tolerances of biotic and abiotic stress in plants. However, the mechanisms mediated by these STS genes remain unclear. To provide insight into the role of STS genes defense against biotic and abiotic stress, we overexpressed VqSTS36 in Arabidopsis thaliana and tomato (Micro-Tom) via Agrobacterium -mediated transformation. VqSTS36 -transformed Arabidopsis lines displayed an increased resistance to powdery mildew, but both VqSTS36 -transformed Arabidopsis and tomato lines showed the increased susceptibility to Botrytis cinerea . Besides, transgenic Arabidopsis lines were found to confer tolerance to salt and drought stress in seed and seedlings. When transgenic plants were treated with a different stress, qPCR assays of defense-related genes in transgenic Arabidopsis and tomato suggested that VqSTS36 played a specific role in different phytohormone-related pathways, including salicylic acid, jasmonic acid, and abscisic acid signaling pathways. All of these results provided a better understanding of the mechanism behind the role of VqSTS36 in biotic and abiotic stress. more...

This work aims to give more insight into mechanisms of action of cadmium (Cd) on germinating pea seeds (Pisum sativum L. var. douce province), specifically the different ways by which Cd cations may interfere with the principal factors involved during germination process, notably storage proteins mobilization, amino acids freeing and proteolytic activities. Obtained results revealed that the process of hydrolysis of main storage proteins showed a significant disruption, which resulted in the decrease of the release of free amino acids, thus imposing a lack in nitrogen supply of essential nutrients to growing embryo under Cd stress. This hypothesis was evidenced by Cd-induced changes occurring in main purified protein fractions; Albumins, Legumins and Vicilins, during their breakdown. Besides, at enzymatic level, the activities of main proteases responsible for this hydrolysis were altered. Indeed, assays using synthetic substrates and specific protease inhibitors followed by protease activity measurements demonstrated that Cd inhibited drastically the total azocaseinolytic activity (ACA) and activities of different proteolytic classes: cysteine-, aspartic-, serine- and metallo-endopeptidases (EP), leucine- and proline-aminopeptidases (LAP and PAP, respectively), and glycine-carboxypeptidases (Gly-CP). The data here presented may suggest that the vulnerability of the embryonic axes towards Cd toxicity could be explained as a result of eventual disruption of metabolic pathways that affect mobilization of reserves and availability of nutrients. In vitro studies suggest that Cd cations may act either directly on the catalytic sites of the proteolytic enzymes, which may cause their deactivation, or indirectly via the generation of oxidative stress and overproduction of free radicals that can interact with enzymes, by altering their activity and structure., (Copyright © 2018 Elsevier GmbH. All rights reserved.) more...

Arctium lappa L. (Burdock) is an important plant with various pharmacological effects. According to the importance of this plant, optimization of its tissue culture will lead to more investigation and application of it. The aim of this study was to develop protocols for callus induction and shoot regeneration of A.  lappa. In order to optimize of tissue culture in A. lappa, callus induction, indirect regeneration and direct regeneration were carried out in factorial experiment based on Completely Randomized Designs (CRDs). Hypocotyl and cotyledon were cultured on the Murashige and Skoog (MS) medium supplemented with different concentrations and combinations of 2,4-Dichlorophenoxyacetic acid (2,4-D) and 6-Benzylaminopurine (BAP) for callus induction. In indirect regeneration experiment various levels of BAP and α-Naphthaleneacetic acid (NAA) and two types of explants (calli derived from cotyledon and hypocotyl) were investigated. In direct regeneration section, various levels of BAP plus 2 mg/l NAA and different explants (cotyledon, hypocotyl and bud) were compared. In both cotyledon and hypocotyl, the maximum callus induction was observed on a media containing 2 mg/l 2,4-D plus 1 mg/l BAP (100% and 76.19% respectively). The highest percentage of indirect regeneration (65%) was observed at 1 mg/l BAP plus 0.5 mg/l NAA on calli from hypocotyl. The highest percentage of direct regeneration (90.33) was observed in hypocotyl with a lateral bud explant on MS medium supplemented with 0.5 mg/l BAP plus 2 mg/l NAA. In this study, optimization of tissue culture protocol for A. lappa was carried out as a research technique, as well as technique for further exploitation of this plant. more...

Nitric oxide (NO) and various reactive nitrogen species produced in cells in normal growth conditions, and their enhanced production under stress conditions are responsible for a variety of biochemical aberrations. The present findings demonstrate that sunflower seedling roots exhibit high sensitivity to salt stress in terms of nitrite accumulation. A significant reduction in S-nitrosoglutathione reductase (GSNOR) activity is evident in response to salt stress. Restoration of GSNOR activity with dithioerythritol shows that the enzyme is reversibly inhibited under conditions of 120 mM NaCl. Salt stress-mediated S-nitrosylation of cytosolic proteins was analyzed in roots and cotyledons using biotin-switch assay. LC-MS/MS analysis revealed opposite patterns of S-nitrosylation in seedling cotyledons and roots. Salt stress enhances S-nitrosylation of proteins in cotyledons, whereas roots exhibit denitrosylation of proteins. Highest number of proteins having undergone S-nitrosylation belonged to the category of carbohydrate metabolism followed by other metabolic proteins. Of the total 61 proteins observed to be regulated by S-nitrosylation, 17 are unique to cotyledons, 4 are unique to roots whereas 40 are common to both. Eighteen S-nitrosylated proteins are being reported for the first time in plant systems, including pectinesterase, phospholipase d-alpha and calmodulin. Further physiological analysis of glyceraldehyde-3-phosphate dehydrogenase and monodehydroascorbate reductase showed that salt stress leads to a reversible inhibition of both these enzymes in cotyledons. However, seedling roots exhibit enhanced enzyme activity under salinity stress. These observations implicate the role of S-nitrosylation and denitrosylation in NO signaling thereby regulating various enzyme activities under salinity stress in sunflower seedlings., (© 2017 Scandinavian Plant Physiology Society.) more...

Key Message: The Coffea arabica BURP domain-containing gene plays an important role in the response of transgenic Arabidopsis plants to abiotic stresses via regulating the level of diverse proteins. Although the functions of plant-specific BURP domain-containing proteins (BDP) have been determined for a few plants, their roles in the growth, development, and stress responses of most plant species, including coffee plant (Coffea arabica), are largely unknown. In this study, the function of a C. arabica BDP, designated CaBDP1, was investigated in transgenic Arabidopsis plants. The expression of CaBDP1 was highly modulated in coffee plants subjected to drought, cold, salt, or ABA. Confocal analysis of CaBDP1-GFP fusion proteins revealed that CaBDP1 is localized in the endoplasmic reticulum. The ectopic expression of CaBDP1 in Arabidopsis resulted in delayed germination of the transgenic plants under abiotic stress and in the presence of ABA. Cotyledon greening and seedling growth of the transgenic plants were inhibited in the presence of ABA due to the upregulation of ABA signaling-related genes like ABI3, ABI4, and ABI5. Proteome analysis revealed that the levels of several proteins are modulated in CaBDP1-expressing transgenic plants. The results of this study underscore the importance of BURP domain proteins in plant responses to diverse abiotic stresses. more...

In agriculture, heavy metal contamination of soil interferes with processes associated with plant growth, development and productivity. Here, we describe oxidative and redox changes, and deleterious injury within cotyledons and seedlings caused by exposure of germinating (Phaseolus vulgaris L. var. soisson nain hâtif) seeds to copper (Cu). Cu induced a marked delay in seedling growth, and was associated with biochemical disturbances in terms of intracellular oxidative status, redox regulation and energy metabolism. In response to these alterations, modulation of activities of antioxidant proteins (thioredoxin and glutathione reductase, peroxiredoxin) occurred, thus preventing oxidative damage. In addition, oxidative modification of proteins was detected in both cotyledons and seedlings by one- and two-dimensional electrophoresis. These modified proteins may play roles in redox buffering. The changes in activities of redox proteins underline their fundamental roles in controlling redox homeostasis. However, observed differential redox responses in cotyledon and seedling tissues showed a major capacity of the seedlings' redox systems to protect the reduced status of protein thiols, thus suggesting quantitatively greater antioxidant protection of proteins in seedlings compared to cotyledon. To our knowledge, this is the first comprehensive redox biology investigation of the effect of Cu on seed germination. more...

Concepts of organelle-to-nucleus signalling pathways are largely based on genetic screens involving inhibitors of chloroplast and mitochondrial functions such as norflurazon, lincomycin (LINC), antimycin A (ANT) and salicylhydroxamic acid. These inhibitors favour enhanced cellular oxidation, but their precise effects on the cellular redox state are unknown. Using the in vivo reduction-oxidation (redox) reporter, roGFP2, inhibitor-induced changes in the glutathione redox potentials of the nuclei and cytosol were measured in Arabidopsis thaliana root, epidermal and stomatal guard cells, together with the expression of nuclear-encoded chloroplast and mitochondrial marker genes. All the chloroplast and mitochondrial inhibitors increased the degree of oxidation in the nuclei and cytosol. However, inhibitor-induced oxidation was less marked in stomatal guard cells than in epidermal or root cells. Moreover, LINC and ANT caused a greater oxidation of guard cell nuclei than the cytosol. Chloroplast and mitochondrial inhibitors significantly decreased the abundance of LHCA1 and LHCB1 transcripts. The levels of WHY1 , WHY3 and LEA5 transcripts were increased in the presence of inhibitors. Chloroplast inhibitors decreased AOXA1 mRNA levels, while mitochondrial inhibitors had the opposite effect. Inhibitors that are used to characterize retrograde signalling pathways therefore have similar general effects on cellular redox state and gene expression.This article is part of the themed issue 'Enhancing photosynthesis in crop plants: targets for improvement'., (© 2017 The Authors.) more...

Sodium nitroprusside (SNP), diethylenetriamine NONOate (DETA), S-nitroso-n-acetyl-D,L- penicillamine (SNAP), and 4-(p-methoxyphenyl)-1,3,2- Oxathiazolylium-5-olate (CAY) exhibit differential NO releasing ability in aqueous solution and hemoglobin is a more efficient NO quencher than cPTIO in solution. DETA releases 16% more NO compared with SNP in solution. Various NO donors (SNP, DETA, SNAP, and CAY) also bring about a differential but concentration-dependent increase in endogenous NO in seedling cotyledons and roots. Two-day old, dark-grown seedling roots exhibit 95%, 77%, 59% and 45% increase in NO content in presence of each of 500 µM of DETA, SNAP, CAY and SNP, respectively, relative to control. NO accumulation in the tissue system as a response to NO donors is reflected in terms of corresponding peroxynitrite accumulation. Release of cyanide and free iron as byproducts of SNP dissociation in solution limits its usefulness as an NO donor. SNP leads to profuse ROS generation in sunflower seedling roots. Light is not a pre-requisite for NO generation from SNP. Present work also demonstrates the usefulness of hemoglobin over cPTIO as NO scavenger. Hemoglobin brings about increasing NO quenching with its increasing concentration from 2.5 to 10 µM. Greater sensitivity of the root system to the NO donor/scavenger treatments is evident, it being in direct contact with the molecules in the incubation/ growth medium. This differential effect does not seem to be significantly transmitted to the cotyledons (long-distance signaling). more...

Plant-derived smoke - butenolide, called at present karrikin 1 (KAR 1 ) is known as an important inductor of seed germination and seedling growth. In this study, tricyclic butenolides were synthesized and their effects on germination of dormant and non-dormant Avena fatua caryopses were compared, as were also their effects versus those of KAR 1 on seedling growth. KAR 1 was found to be most effective and to completely remove dormancy. Butenolides, rac-8 and (S)-8a, showed a low stimulatory effect on germination of dormant caryopses, visible only when applied at very high concentrations. These compounds used at concentrations 100 times those of KAR 1 similarly increased the speed of germination and vigor of non-dormant caryopses. Likewise, growth of coleoptiles and their fresh weight were increased by KAR 1 as well as by rac-8 and (S)-8a to a similar value. KAR 1 and rac-8 were more effective than (S)-8a in increasing root growth. The results shown indicate that the presence of an aromatic ring in the absence of methyl group at C3 induced a much lower, or a similar, effect on germination of dormant and non-dormant Avena fatua caryopses and seedling growth compared to KAR 1 , but only when used at much higher concentrations. The simultaneous presence of a methyl group at C3 and an aromatic ring in the compound rac-7 exerted only a slight effect on the root growth., (Copyright © 2017 Elsevier GmbH. All rights reserved.) more...

A germinating seedling undergoes skotomorphogenesis to emerge from the soil and reach for light. During this phase, the cotyledons are closed, and the hypocotyl elongates. Upon exposure to light, the seedling rapidly switches to photomorphogenesis by opening its cotyledons and suppressing hypocotyl elongation. The E3 ubiquitin ligase CONSTITUTIVE PHOTOMORPHOGENIC 1 (COP1) is critical for maintaining skotomorphogenesis. Here, we report that jasmonate (JA) suppresses hypocotyl elongation and stimulates cotyledon opening in etiolated seedlings, partially phenocopying cop1 mutants in the dark. We also find that JA stabilizes several COP1-targeted transcription factors in a COP1-dependent manner. RNA-seq analysis further defines a JA-light co-modulated and cop1-dependent transcriptome, which is enriched for auxin-responsive genes and genes participating in cell wall modification. JA suppresses COP1 activity through at least two distinct mechanisms: decreasing COP1 protein accumulation in the nucleus; and reducing the physical interaction between COP1 and its activator, SUPPRESSOR OF PHYTOCHROME A-105 1 (SPA1). Our work reveals that JA suppresses COP1 activity to stabilize COP1 targets, thereby inhibiting hypocotyl elongation and stimulating cotyledon unfolding in etiolated Arabidopsis seedlings., (© 2017 The Authors The Plant Journal © 2017 John Wiley & Sons Ltd.) more...

Artificial excision of the distal part of acorns in order to promote germination is well researched in oak seedling cultivation studies. However, studies of combined effects of cotyledon excision and nursery fertilization on container seedlings are lacking, especially for seedling root growth and outplanting performance. This study aimed to explore the main effects of cotyledon excision on Quercus variabilis seedling emergence characteristics and demonstrated the combined effects of cotyledon excision and nursery fertilization on seedling quality to improve Quercus variabilis seedling outplanting performance. Four cotyledon excision treatments and two classes of nursery fertilization were implemented. Seedling emergence was noted every week after sowing. Seedling dry mass, morphology, and nutrient status were assessed at the end of the nursery season. After the first outplanting season, the aforementioned measurements along with seedling survival were determined once again. The results showed that cotyledon excision generally induced greater and more rapid seedling emergence, but did not affect shoot emergence synchronicity. The highest total emergence and emergence rate occurred with Intermediate excision (1/2 of the distal end of acorn was excised). Effects of nutrient loss due to cotyledon excision on seedling quality and outplanting performance were somewhat compensated by nursery fertilization. Nursery fertilization promoted dry mass increment (the net increment from T0 to T2 for dry mass) for excised seedlings after outplanting, resulting in better performance for Slight (1/3 of the distal end of acorn was excised) and Intermediate excision treatments in the field. Thus we conclude Intermediate excision combined with reasonable nursery fertilization can be recommended for production of nursery grown seedlings for afforestation. more...

Elongation of the mesocotyl and coleoptile facilitates the emergence of rice ( Oryza sativa ) seedlings from soil and is affected by various genetic and environment factors. The regulatory mechanism underlying this process remains largely unclear. Here, we examined the regulation of mesocotyl and coleoptile growth by characterizing a gaoyao1 ( gy1 ) mutant that exhibits a longer mesocotyl and longer coleoptile than its original variety of rice. GY1 was identified through map-based cloning and encodes a PLA 1 -type phospholipase that localizes in chloroplasts. GY1 functions at the initial step of jasmonic acid (JA) biosynthesis to repress mesocotyl and coleoptile elongation in etiolated rice seedlings. Ethylene inhibits the expression of GY1 and other genes in the JA biosynthesis pathway to reduce JA levels and enhance mesocotyl and coleoptile growth by promoting cell elongation. Genetically, GY1 acts downstream of the OsEIN2-mediated ethylene signaling pathway to regulate mesocotyl/coleoptile growth. Through analysis of the resequencing data from 3000 rice accessions, we identified a single natural variation of the GY1 gene, GY1 376T , which contributes to mesocotyl elongation in rice varieties. Our study reveals novel insights into the regulatory mechanism of mesocotyl/coleoptile elongation and should have practical applications in rice breeding programs., (© 2017 American Society of Plant Biologists. All rights reserved.) more...

Chili pepper (Capsicum annuum L.) ranks among the most important vegetable crop belonging to the family Solanaceae that is consumed both as vegetable and spice throughout the world. C. annuum, as crop, in order to meet the target yield, demands improved variety that could overcome environmental challenges viz., biotic and abiotic stress. Cultivar improvement essentially requires an efficient in vitro regeneration protocol. In the present study, we investigated the influence of silver nitrate (AgNO3) and coconut water, individually as well in combination, on in vitro shoot elongation and plant regeneration from cotyledon explants of C. annuum cv G-4. Shoot buds were induced on shoot bud induction medium supplemented with either 44.38 µM 6-benzylaminopurine (BAP) or 9.0 µM thidiazuron (TDZ) along with 5.77 µM gibberellic acid (GA3) and 14.7 µM phenyl acetic acid (PAA). Elongation of shoot buds was obtained on elongation medium containing 8.87 µM BA or 0.45 µM TDZ, 5.77 µM GA3 and 14.7 µM PAA followed by rooting in 9.8 µM indole-3-butyric acid (IBA). All the media were supplemented with 30 µM AgNO3 and/or coconut water (10% v/v). The presence of coconut water in the elongation media enhanced the regeneration of well developed shoots from differentiating explants on TDZ media while AgNO3 resulted in enhanced production of rooted shoots with greater influence on emerging shoots from BAP media upon transfer to rooting media. There was synergistic response with further enhancement of elongated shoots/elongated rooted shoots on the combined use of coconut water and AgNO3. The elongation media produced significantly higher total shoots when AgNO3 was used synergistically with coconut water (59.0%) as against AgNO3 alone (38.0%). While in rooting media, there was significantly higher production of elongated rooted shoots when coconut water was used synergistically with AgNO3 (47.2%) as against the coconut water alone (14.4%). more...

Identification of related mutants contributed a lot to our understanding of the molecular mechanisms underlying brassinosteroid (BR) signaling. Establishment of methods to test for the hormone sensitivity is essential for the characterization of the mutants associated with BR responses. Here, we describe detailed procedures for the physiological analysis of BR responses that are regularly applied to test the BR sensitivity of mutants by evaluating the growth of different tissues of rice (Oryza sativa) seedlings. more...

When wounding or grafting interrupts the original connection of plant tissue, cell proliferation is induced and the divided tissue is reunited. Previous studies suggested that gibberellin derived from the cotyledon is required for tissue reunion in cucumber and tomato incised hypocotyls, and tissue reunion of Arabidopsis incised flowering stems is controlled by auxin. Differences in the hormone requirements of the tissue reunion process between Arabidopsis and cucumber might be due to differences in organs or species. In this study, we performed morphological and gene expression analyses of graft union in Arabidopsis hypocotyl. We found that removal of the cotyledon and treatment of the cotyledon with the auxin transport inhibitor triiodobenzoic acid (TIBA) suppressed cell proliferation of vascular tissue during graft union formation. These treatments also suppressed expression of IAA5, ANAC071, ANAC096 and CYCB1;1. ANAC071 is involved in the tissue reunion process. The anac071 anac096 double mutant suppressed cell proliferation more so than either of the single mutants. On the other hand, paclobutrazol treatment or deficiency of gibberellin biosynthesis genes suppressed expansion of cortex cells, and exogenous gibberellin treatment or rga/gai mutations that lack the negative regulator of gibberellin reversed this inhibition. The up-regulation of the key gibberellin biosynthesis gene GA20ox1 during graft union formation was prevented by cotyledon removal or TIBA treatment. These data suggest that auxin regulates cell proliferation of vascular tissue and expansion of cortex cells by promoting gibberellin biosynthesis during graft attachment. We hypothesize that the cotyledon-derived phytohormones are essential for graft reunion of the hypocotyl, processed in a cell type-specific manner, in Arabidopsis., (© The Author 2016. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please email: journals.permissions@oup.com.) more...

Key Message: Enhancement of endoreduplication in dark-grown hypocotyl is a common feature in dicotyledonous polysomatic plants, and TIBA-mediated inhibition of the endoreduplication is partially due to abnormal actin organization. Many higher plant species use endoreduplication during cell differentiation. However, the mechanisms underlying this process have remained elusive. In this study, we examined endoreduplication in hypocotyls and cotyledons in response to light in some dicotyledonous plant species. Enhancement of endoreduplication was found in the dark-grown hypocotyls of all the polysomatic species analyzed across five different families, indicating that this process is a common feature in dicotyledonous plants having polysomatic tissues. Conversely, endoreduplication was enhanced in the light-grown cotyledons in four of the five species analyzed. We also analyzed the effect of a polar auxin transport inhibitor, 2,3,5-triiodobenzoic acid (TIBA) on endoreduplication in hypocotyl and cotyledon tissues of radish (Raphanus sativus L. var. longipinnatus Bailey). TIBA was found to inhibit and promote endoreduplication in hypocotyls and cotyledons, respectively, suggesting that the endoreduplication mechanism differs in these organs. To gain insight into the effect of TIBA, radish and spinach (Spinacia oleracea L.) seedlings were treated with a vesicle-trafficking inhibitor, brefeldin A, and an actin polymerization inhibitor, cytochalasin D. Both of the inhibitors partially inhibited endoreduplication of the dark-grown hypocotyl tissues, suggesting that the prominent inhibition of endoreduplication by TIBA might be attributed to its multifaceted role. more...

The toxicity of azetidine-2-carboxylic acid (A2C), a structural analogue of L-proline, results from its incorporation into proteins due to misrecognition by prolyl-tRNA synthetase (ProRS). The growth of Arabidopsis thaliana seedling roots is more sensitive to inhibition by A2C than is cotyledon growth. Arabidopsis contains two ProRS isozymes. AtProRS-Org (At5g52520) is localized in chloroplasts/mitochondria, and AtProRS-Cyt (At3g62120) is cytosolic. AtProRS-Cyt mRNA is more highly expressed in roots than in cotyledons. Arabidopsis ProRS isoforms were expressed as His-tagged recombinant proteins in Escherichia coli. Both enzymes were functionally active in ATP-PPi exchange and aminoacylation assays, and showed similar K m for L-proline. A major difference was observed in the substrate specificity of the two enzymes. AtProRS-Cyt showed nearly identical substrate specificity for L-proline and A2C, but for AtProRS-Org the specificity constant was 77.6 times higher for L-proline than A2C, suggesting that A2C-sensitivity may result from lower amino acid specificity of AtProRS-Cyt. Molecular modelling and simulation results indicate that this specificity difference between the AtProRS isoforms may result from altered modes of substrate binding. Similar kinetic results were obtained with the ProRSs from Zea mays, suggesting that the difference in substrate specificity is a conserved feature of ProRS isoforms from plants that do not accumulate A2C and are sensitive to A2C toxicity. The discovery of the mode of action of A2C toxicity could lead to development of biorational weed management strategies., (© 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.) more...

The effect of pulsed magnetic field (PMF) on gravitropic response, endogenous growth and growth in the presence of indole-3-acetic acid (IAA) was studied in coleoptiles of maize (Zea mays L.) seedlings. Medium pH changes, measured simultaneously with growth of coleoptile segments, were also determined. In seedlings grown in the presence of PMF, elongation growth of coleoptiles was inhibited by 16%, while growth of roots and mesocotyls did not depend on PMF. Magnetic field also inhibited (by 36%) the gravitropic response of maize seedlings. However, when PMF was applied only during gravistimulation (within 6 h), it suppressed the gravitropic reaction only by 8% at 6 h. It was also found that endogenous growth and IAA-induced growth of maize coleoptile segments excised from seedlings treated with the PMF was stimulated by 52% and 30%, respectively, as compared to control (segments untreated with the PMF). Values of medium pH, measured simultaneously with growth, indicated that PMF-treated coleoptile segments extruded much more protons than untreated segments. In contrast, coleoptile segments treated with the PMF and subsequently incubated in the presence of IAA extruded 2.5-fold less protons as compared to segments treated with IAA only. The data presented here have been discussed with consideration of mechanisms by which auxin (IAA) regulates plant cell growth. more...

The present findings demonstrate significant modulation of total glutathione content, reduced glutathione (GSH) content, oxidized glutathione (GSSG) content, GSH/GSSG ratio and glutathione reductase (GR; EC 1.6.4.2) activity in dark-grown seedling cotyledons in response to salt-stress (120 mM NaCl) in sunflower (Helianthus annuus L.) seedlings. A differential spatial distribution of GR activity (monitored by confocal laser scanning microscopic (CLSM) imaging) is also evident. Melatonin and nitric oxide (NO) differentially ameliorate salt stress effect by modulating GR activity and GSH content in seedling cotyledons. Total glutathione content (GSH + GSSG) exhibit a seedling age-dependent increase in the cotyledons, more so in salt-stressed conditions and when subjected to melatonin treatment. Seedlings raised in presence of 15 μM of melatonin exhibit significant increase in GR activity in cotyledon homogenates (10,000 g supernatant) coinciding with significant increase in GSH content. GSSG content and GSH/GSSG ratio also increased due to melatonin treatment. A correlation is thus evident in NaCl-sensitized modulation of GSH content and GR activity by melatonin. GSH content is down regulated by NO provided as 250 μM of sodium nitroprusside (SNP) although total glutathione content remained in similar range. A reversal of response (enhanced total glutathione accumulation) by NO scavenger (cPTIO) highlights the critical role of NO in modulating glutathione homeostasis. SNP lowers the activity of hydroxyindole-O-methyltransferase (HIOMT) - a regulatory enzyme in melatonin biosynthesis in control seedlings whereas its activity is upregulated in salt-stressed seedling cotyledons. Melatonin content of seedling cotyledons is also modulated by NO. NO and melatonin thus seem to modulate GR activity and GSH content during seedling growth under salt stress., (Copyright © 2016 Elsevier Inc. All rights reserved.) more...

Ten agronomic plant species were exposed to different concentrations of nano-titanium dioxide (nTiO2 ) or nano-cerium oxide (nCeO2 ) (0 μg/mL, 250 μg/mL, 500 μg/mL, and 1000 μg/mL) to examine potential effects on germination and early seedling development. The authors modified a standard test protocol developed for soluble chemicals (OPPTS 850.4200) to determine if such an approach might be useful for screening engineered nanomaterials (ENMs) and whether there were differences in response across a range of commercially important plant species to 2 common metal oxide ENMs. Eight of 10 species responded to nTiO2 , and 5 species responded to nCeO2 . Overall, it appeared that early root growth may be a more sensitive indicator of potential effects from ENM exposure than germination. The observed effects did not always relate to the exposure concentration, indicating that mass-based concentration may not fully explain the developmental effects of these 2 ENMs. The results suggest that nTiO2 and nCeO2 have different effects on early plant growth of agronomic species, with unknown effects at later stages of the life cycle. In addition, standard germination tests, which are commonly used for toxicity screening of new materials, may not detect the subtle but potentially more important changes associated with early growth and development in terrestrial plants. Environ Toxicol Chem 2016;35:2223-2229. Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America., (Published 2016 Wiley Periodicals Inc. on behalf of SETAC. This article is a US Government work and, as such, is in the public domain in the United States of America.) more...

The work described herein is a continuation of a previous study centered on the bioprospect of cardoon (Cynara cardunculus) leaf extracts through the isolation of secondary metabolites with phytotoxic activity. Chromatographic fractionations of the ethyl acetate extract and spectroscopic analysis showed that the majority of the components were sesquiterpene lactones. Of these compounds, aguerin B, grosheimin, and cynaropicrin were very active on etiolated wheat coleoptile, standard target species, and weed growth. The joint action of binary mixtures of these three active sesquiterpene lactones and one nonactive compound (11,13-dihydroxy-8-desoxygrosheimin) was studied. The activities of fixed-ratio mixtures were assessed on wheat coleoptile. The results can be interpreted with respect to a reference model by considering dose-response analyses and isobolograms with linear regression analyses. A total of 17 binary mixtures at different levels of inhibition (ED25, ED50, and ED75) were studied, and predominantly they responded additively (25). Deviations from additivity included seven synergistic responses and two antagonistic responses. The joint action of major sesquiterpene lactones isolated from C. cardunculus can explain the activities observed in extracts and fractions. The results reported here reiterate the utility of the wheat coleoptile bioassay as a quick tool to detect potential synergistic effects in binary mixtures. more...

Despite the increase in recent decades in herbicide research on the potential of native plants, current knowledge is considered to be low. Very few studies have been carried out on the chemical profile or the biological activity of the Brazilian savanna (Cerrado) species. In the study reported here, the allelopathic activity of AcOEt and MeOH extracts of leaves, stems, and roots from Ocotea pulchella Nees was evaluated. The extracts were assayed on etiolated wheat coleoptiles. The AcOEt leaf extract was the most active and this was tested on standard target species (STS). Lycopersicon esculentum and Lactuca sativa were the most sensitive species in this test. A total of eleven compounds have been isolated and characterized. Compounds 1, 2, 4, and 6 have not been identified previously from O. pulchella and ocoteol (9) is reported for the first time in the literature. Eight compounds were tested on wheat coleoptile growth, and spathulenol, benzyl salicylate, and benzyl benzoate showed the highest activities. These compounds showed inhibitory activity on L. esculentum. The values obtained correspond to the activity exhibited by the extract and these compounds may therefore be responsible for the allelopathic activity shown by O. pulchella., (© 2016 Wiley-VHCA AG, Zürich.) more...

To achieve host colonization, successful pathogens need to overcome plant basal defences. For this, (hemi)biotrophic pathogens secrete effectors that interfere with a range of physiological processes of the host plant. AvrLm4-7 is one of the cloned effectors from the hemibiotrophic fungus Leptosphaeria maculans 'brassicaceae' infecting mainly oilseed rape (Brassica napus). Although its mode of action is still unknown, AvrLm4-7 is strongly involved in L. maculans virulence. Here, we investigated the effect of AvrLm4-7 on plant defence responses in a susceptible cultivar of B. napus. Using two isogenic L. maculans isolates differing in the presence of a functional AvrLm4-7 allele [absence ('a4a7') and presence ('A4A7') of the allele], the plant hormone concentrations, defence-related gene transcription and reactive oxygen species (ROS) accumulation were analysed in infected B. napus cotyledons. Various components of the plant immune system were affected. Infection with the 'A4A7' isolate caused suppression of salicylic acid- and ethylene-dependent signalling, the pathways regulating an effective defence against L. maculans infection. Furthermore, ROS accumulation was decreased in cotyledons infected with the 'A4A7' isolate. Treatment with an antioxidant agent, ascorbic acid, increased the aggressiveness of the 'a4a7' L. maculans isolate, but not that of the 'A4A7' isolate. Together, our results suggest that the increased aggressiveness of the 'A4A7' L. maculans isolate could be caused by defects in ROS-dependent defence and/or linked to suppressed SA and ET signalling. This is the first study to provide insights into the manipulation of B. napus defence responses by an effector of L. maculans., (© 2015 BSPP AND JOHN WILEY & SONS LTD.) more...

Biochemical, genetic, physiological, and molecular research in plants has demonstrated a central role of glucose (Glc) in the control of plant growth, metabolism, and development, and has revealed networks that integrate light, stresses, nutrients, and hormone signaling. Previous studies have reported that AtPGR protein as potential candidates for Glc signaling protein. In the present study, we characterized transcription factors that bind to the upstream region of the AtPGR gene isolated using the yeast one-hybrid screening with an Arabidopsis cDNA library. One of the selected genes (AtSTKL) appeared to confer elevated sensitivity to Glc response. Overexpression of AtSTKLs (AtSTKL1 and AtSTKL2) increased the sensitivity to Glc during the post-germination stages. In contrast, atstkl1 and atstkl2 antisense lines displayed reduced sensitivity to high Glc concentration during the early seedling stage. Furthermore, we showed that the two AtSTKLs bind to the 5'-GCCT-3' element of the upstream promoter region of the AtPGR gene in vitro and repress the beta-glucuronidase (GUS) activity in AtPGR promoter-GUS (P999-GUS) transgenic plants. Green fluorescent protein (GFP)-tagged AtSTKLs were localized in the nuclei of transgenic Arabidopsis cells. Collectively, these results suggest that AtSTKL1 and AtSTKL2 function both as repressors of AtPGR transcription and as novel transcription factors in the Glc signaling pathway., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.) more...

During soybean (Glycine max (L.) Merrill) seed development, protease C1, the proteolytic enzyme that initiates breakdown of the storage globulins β-conglycinin and glycinin at acidic pH, is present in the protein storage vacuoles (PSVs), the same subcellular compartments in seed cotyledons where its protein substrates accumulate. Actual proteolysis begins to be evident 24 h after seed imbibition, when the PSVs become acidic, as indicated by acridine orange accumulation visualized by confocal microscopy. Imidodiphosphate (IDP), a non-hydrolyzable substrate analog of proton-translocating pyrophosphatases, strongly inhibited acidification of the PSVs in the cotyledons. Consistent with this finding, IDP treatment inhibited mobilization of β-conglycinin and glycinin, the inhibition being greater at 3 days compared to 6 days after seed imbibition. The embryonic axis does not appear to play a role in the initial PSV acidification in the cotyledon, as axis detachment did not prevent acridine orange accumulation three days after imbibition. SDS-PAGE and immunoblot analyses of cotyledon protein extracts were consistent with limited digestion of the 7S and 11S globulins by protease C1 starting at the same time and proceeding at the same rate in detached cotyledons compared to cotyledons of intact seedlings. Embryonic axis removal did slow down further breakdown of the storage globulins by reactions known to be catalyzed by protease C2, a cysteine protease that normally appears later in seedling growth to continue the storage protein breakdown initiated by protease C1., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.) more...