The Effects of Melatonin on Transcriptional Profile of Unfolded Protein Response Genes Under Endoplasmic Reticulum Stress in <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

When the load of secretory pathway is increased or folding capacity in the endoplasmic reticulum (ER) is insufficient, unfolded proteins might accumulate in ER lumen causing a phenomenon called ER stress. During ER stress, normal cell functions are suppressed and unfolded protein response (UPR) is induced. Studies in animal systems suggest that melatonin alleviates the detrimental effects of ER stress; however, there is no study in plants in this respect. Hence, in this study, we investigated the possible role of melatonin on alleviation of ER stress in model plant Arabidopsis thaliana. Tunicamycin (Tm) was used to specifically induce ER stress. Melatonin treatment (10 and 25 μM but not 1 μM) increased root growth under Tm treatment, but it did not reach control levels. ER stress induced the expressions of ER stress sensor/transducer genes, ER chaperones and folding helper genes, ER-associated degradation (ERAD) genes, and ER stress-associated apoptosis genes in roots and shoots (a total of 16 genes). Among them, the expressions of ER stress sensor/transducer bZIP17, bZIP28, IRE1A, IRE1B, ERAD-related SEL1, and apoptosis genes AGB1 were decreased back to control levels with 25 μM melatonin under ER stress in roots. Moreover, Tm?+?melatonin treatments decreased the expressions of these genes when compared to only Tm-treated plants. Downregulation of UPR components with increased concentrations of melatonin under Tm treatment demonstrated that melatonin alleviated the detrimental effects of ER stress.     

Genome-wide identification and expression analysis of the GRAS family proteins in <Emphasis Type="Italic">Medicago truncatula</Emphasis>

The GRAS gene family performs a variety of functions in plant growth and development processes, and they also play essential roles in plant response to environmental stresses. Medicago truncatula is a diploid plant with a small genome used as a model organism. Despite the vital role of GRAS genes in plant growth regulation, few studies on these genes in M. truncatula have been conducted to date. Using the M. truncatula reference genome data, we identified 68 MtGRAS genes, which were classified into 16 groups by phylogenetic analysis, located on eight chromosomes. The structure analysis indicated that MtGRAS genes retained a relatively constant exon–intron composition during the evolution of the M. truncatula genome. Most of the closely related members in the phylogenetic tree had similar motif compositions. Different motifs distributed in different groups of the MtGRAS genes were the sources of their functional divergence. Twenty-eight MtGRAS genes were expressed in six tissues, namely root, bud, blade, seedpod, nodule, and flower tissues, suggesting their putative function in many aspects of plant growth and development. Nine MtGRAS genes were upregulated under cold, freezing, drought, ABA, and salt stress treatments, indicating that they play vital roles in the response to abiotic stress in M. truncatula. Our study provides valuable information that can be utilized to improve the quality and agronomic benefits of M. truncatula and other plants.     

<Emphasis Type="Italic">In vitro</Emphasis> growth profile and comparative leaf anatomy of the C<Subscript>3</Subscript>–C<Subscript>4</Subscript> intermediate plant <Emphasis Type="Italic">Mollugo nudicaulis</Emphasis> Lam.

Mollugo nudicaulis Lam., commonly known as John’s folly or naked-stem carpetweed, is an ephemeral species of tropical regions. The plant is ideal to study the eco-physiological adaptations of C₃–C₄ intermediate plants. In the present report, in vitro growth profiling of the plant and comparative leaf anatomy under in vitro and ex vitro conditions were studied. In vitro propagation of the plant was carried out on Murashige and Skoog (MS) basal medium augmented with additives and solidified with 0.8% (w/v) agar-agar or 0.16% (w/v) Phytagel?. The concentration of plant growth regulators (PGRs) in the basal medium was optimized for callus induction, callus proliferation, shoot regeneration, and in vitro rooting. The optimum callus induction was obtained from M. nudicaulis seedling hypocotyls. The highest regeneration induction of about 88% or nearly 41 shoots with about 142 leaves per culture vessel was observed from friable callus on MS basal medium solidified with Phytagel? and containing 4.44 μM 6-benzylaminopurine, 4.65 μM kinetin, 2.69 μM naphthaleneacetic acid, and 0.91 μM thidiazuron. In leaf anatomy, differences related to photosynthetic tissue organization were observed in leaves of in vitro and ex vitro plants, which indicated that changes in the environment affected the anatomy of subsequent leaves in plants. This is the first report of an efficient micropropagation protocol for M. nudicaulis, using an indirect organogenesis method. Efforts were made to optimize the concentrations of various PGRs and organic compounds for in vitro growth of regenerated shoots.     

Cloning and functional analysis of two <Emphasis Type="Italic">GmDeg</Emphasis> genes in soybean [<Emphasis Type="Italic">Glycine max</Emphasis> (L.) Merr.]

Although light is the ultimate substrate in photosynthesis, strong light can also be harmful and lead to photoinhibition. The DEG proteases play important roles in the degradation of misfolded and damaged proteins. In this study, two photoinhibition-related genes from soybean [Glycine max (L.) Merr.], GmDeg1 and GmDeg2, were cloned. Bioinformatics analysis indicated that these two proteases both contain a PDZ domain and are serine proteases. The expression levels of GmDeg1 and GmDeg2 increased significantly after 12 h of photooxidation treatment, indicating that GmDeg1 and GmDeg2 might play protective roles under strong light conditions. In in vitro proteolytic degradation assays, recombinant GmDeg1 and GmDeg2 demonstrated biological activities at temperatures ranging from 20°C to 60°C and at pH 5.0 to 8.0. By contrast, the proteases showed no proteolytic effect in the presence of a serine protease inhibitor. Taken together, these results provided strong evidence that GmDeg1 and GmDeg2 are serine proteases that could degrade the model substrate in vitro, indicating that they might degrade damaged D1 protein and other mis-folded proteins in vivo. Furthermore, GmDeg1 and GmDeg2 were transformed into Arabidopsis thaliana to obtain transgenic plants. Leaves from the transgenic and wild-type plants were subjected to strong light conditions in vitro, and the PSII photochemical efficiency (Fv/Fm) was measured. The Fv/Fm of the transgenic plants was significantly higher than that of the wild-type plants at most time points. These results imply that GmDeg1 and GmDeg2 would have similar functions to Arabidopsis AtDeg1, thus accelerating the recovery of PSII photochemical efficiency.     

Molecular cloning and functional characterization of a Cu/Zn superoxide dismutase gene (<Emphasis Type="Italic">CsCSD1</Emphasis>) from <Emphasis Type="Italic">Cucumis sativus</Emphasis>

Superoxide dismutase (SOD) proteins, which are widely present in the plant kingdom, play vital roles in response to abiotic stress. However, the functions of cucumber SOD genes in response to environmental stresses remain poorly understood. In this study, a SOD gene CsCSD1 was identified and functionally characterized from cucumber (Cucumis sativus). The CsCSD1 protein was successfully expressed in E. coli, and its overexpression significantly improved the tolerance of host E. coli cells to salinity stress. Besides, overexpression of CsCSD1 enhanced salinity tolerance during germination and seedling development in transgenic Arabidopsis plants. Further analyses showed that the SOD and CAT (catalase) activities of transgenic plants were significantly higher than those of wild-type (WT) plants under normal growth conditions as well as under NaCl treatment. In addition, the expression of stress-response genes RD22, RD29B and LEA4-5 was significantly elevated in transgenic plants. Our results demonstrate that the CsCSD1 gene functions in defense against salinity stress and may be important for molecular breeding of salt-tolerant plants.     

UBIQUITIN-SPECIFIC PROTEASES function in plant development and stress responses

Key message

UBIQUITIN-SPECIFIC PROTEASES play important roles in plant development and stress responses.

Abstract

Protein ubiquitination and deubiquitination are reversible processes, which can modulate the stability, activity as well as subcellular localization of the substrate proteins. UBIQUITIN-SPECIFIC PROTEASE (UBP) protein family participates in protein deubiquitination. Members of UBP family are involved in a variety of physiological processes in plants, as evidenced by their functional characterization in model plant Arabidopsis and other plants. UBPs are conserved in plants and distinct UBPs function in different regulatory processes, although functional redundancies exist between some members. Here we briefly reviewed recent advances in understanding the biological functions of UBP protein family in Arabidopsis, particularly the molecular mechanisms by which UBPs regulate plant development and stress responses. We believe that elucidation of UBPs function and regulation in Arabidopsis will provide new insights about protein deubiquitination and might shed light on the understanding of the mechanistic roles of UBPs in general, which will definitely contribute to crop improvement in agriculture.

     

Anatomy and photosystem II activity of <Emphasis Type="Italic">in vitro</Emphasis> grown <Emphasis Type="Italic">Aechmea blanchetiana</Emphasis> as affected by 1-naphthaleneacetic acid

Auxins are one of the main regulators of in vitro plant growth and development. However, the mechanisms, by which auxins, such as 1-naphthaleneacetic acid (NAA), affect in vitro root and leaf anatomy and photosystem function, remain unclear. Accordingly, the aim of the present study was to analyze the effect of different NAA concentrations on the anatomy and photosynthetic performance of in vitro-propagated Aechmea blanchetiana and to determine whether such a treatment affects micropropagated plants after acclimatization. In vitro-established A. blanchetiana plants were transferred to culture media that contained 0, 2, 4, or 6 μM NAA, and after 50 d, they were transplanted into plastic seedling trays with a commercial substrate and cultivated for 60 d in a greenhouse. The plants were evaluated after a 50-d in vitro NAA exposure (growth traits, chlorophyll α fluorescence, and root and leaf anatomy) and after 60 d of acclimatization in the greenhouse (root and leaf growth). Changes induced by NAA in root anatomy might improve uptake of minerals and sugars from the medium, thereby increasing the in vitro growth. In the leaves, the lowest chlorenchyma thickness and sclerenchyma area were observed in plants grown without NAA, and NAA exposure also improved photosystem II activity. The highest ex vitro growth rate was observed for plants that were propagated with 4 μM NAA. Therefore, the use of NAA during in vitro propagation can improve the anatomical and physiological quality of A. blanchetiana plants, as well as to improve ex vitro transfer.     

Native and exotic foundation grasses differ in traits and responses to belowground tri-trophic interactions

A plant’s growth and fitness are influenced by species interactions, including those belowground. In primary successional systems, belowground organisms are known to have particularly important control over plant growth. Exotic plant invasions in these and other habitats may in part be explained by altered associations with belowground organisms compared to native plants. We investigated the growth responses of two foundation grasses on Great Lakes sand dunes, the native grass Ammophila breviligulata and the exotic grass Leymus arenarius, to two groups of soil organisms with important roles in dune succession: arbuscular mycorrhizal fungi (AMF) and plant-parasitic nematodes (PPN). We manipulated the presence/absence of two generalist belowground species known to occur in Great Lakes dunes, Rhizophagus intraradices (AMF) and Pratylenchus penetrans (PPN) in a factorial greenhouse experiment and assessed the biomass production and root architectural traits of the plants. There were clear differences in growth and above- and belowground architecture between Ammophila and Leymus, with Leymus plants being bigger, taller, and having longer roots than Ammophila. Inoculation with Rhizophagus increased above- and belowground biomass production by ~32% for both plant species. Inoculation with Pratylenchus decreased aboveground biomass production by ~36% for both plant species. However belowground, the exotic Leymus was significantly more resistant to PPN than the native Ammophila, and gained more benefits from AMF in belowground tri-trophic interactions than Ammophila. Overall, our results indicate that differences in plant architecture coupled with altered belowground interactions with AMF and PPN have the potential to promote exotic plant invasion.     

One gene member of the ADP-ribosylation factor family is heat-inducible and enhances seed germination in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

ADP ribosylation factors (ARFs), one group within the Ras superfamily of GTP-binding proteins, are ubiquitous within the eukaryotic kingdom. The functions of ARFs are extensive, and include regulatory roles in vesicular transportation, lipid metabolism, and microtubule dynamics, and the cellular processes related to these roles. Most ARFs have been identified from mammalian species and yeast; although little is known about the functional importance of ARFs in plants, it seems to be equally diverse and significant. We have been working on plant responses under heat stress, and showed that heat-shock can induce seed germination (Koo et al. in Plant Physiol 167:1030–1038, 2015). In the present study, we report nine ARF gene family members from tobacco (Nicotiana tabacum), all belonging to the same group (Class 1) in the phylogenetic analysis. One family member, NtARF1, was induced under high-temperature stress. To elucidate the biological function of NtARF1, we generated transgenic tobacco plants overexpressing NtARF1 and the seeds of these transgenic tobacco plants germinated earlier than the seeds of non-transgenic tobacco plants. We also classified ARF family genes in plant species through systematic genomic DNA sequence data-mining, focusing on the fully sequenced and extensively annotated genomes of Arabidopsis thaliana, Brachypodium distachyon, Medicago truncatula, Mimulus guttatus, Nicotiana benthamiana, Setaria italica, Solanum lycopercisum, and Solanum tuberosum, and of some major crops including rice, soybean, corn, and tobacco. The Class 1 of our phylogenetics analysis comprised the highest number of ARFs among the four groups obtained for all plant species analyzed, especially for crop plant species.     

Cryopreservation of the critically endangered golden paintbrush (<Emphasis Type="Italic">Castilleja levisecta</Emphasis> Greenm.): from nature to cryobank to nature

In this study conservation of Castilleja levisecta Greenm., a globally endangered species was addressed through in vitro cryopreservation of shoot tips. In vitro cultures were successfully established using seedlings received from British Columbia, Canada. Shoot tips excised from in vitro propagated plants were cryopreserved using a droplet-vitrification method following optimization of individual protocol steps such as pre-culture, treatment with vitrification solutions, and unloading. The highest plant regrowth after cryopreservation (66%) was achieved when shoot tips were pre-cultured in 0.3 M sucrose for 17 h followed by 0.5 M sucrose for 4 h, incubated in an osmo-protectant solution (17.5% [v/v] glycerol and 17.5% [w/v] sucrose) for 20 min, exposed to vitrification solution A3 (37.5% [v/v] glycerol plus 15% [v/v] dimethylsulfoxide (DMSO) plus 15% [v/v] ethylene glycol (EG) plus 22.5% [w/v] sucrose) on ice for 40 min, and unloaded in 0.8 M sucrose solution for 30 min. Healthy plants were developed from cryopreserved shoot tips and propagated in vitro using nodal segments. Plants derived from in vitro culture and from cryopreserved tissues were successfully rooted and acclimated in a greenhouse with 100% survival rate. Acclimatized plants were reintroduced in a naturalized propagation area at the Conservation Nursery at Fort Rodd Hill, Canada. Twenty of 94 reintroduced plants (21%) survived the transit from lab to field and some had started to flower. This is the first report for cryopreservation of C. levisecta, an important step in conserving and re-introducing this critically imperiled species in nature.     

<Emphasis Type="Italic">In vitro</Emphasis> and <Emphasis Type="Italic">in vivo</Emphasis> antifungal activity of crude extracts and powdered dry material from Ethiopian wild plants against economically important plant pathogens

In Ethiopia extracts from specific plants are used traditionally as natural fungicides in small scale farming systems where synthetic chemicals are out of reach of the average subsistence farmer while no scientific base exists for this practice. Subsequently, methanolic crude extracts from Dolichos kilimandscharicus and Maerua subcordata roots as well as Phytolacca dodecandra berries were screened in vitro for antifungal activity against Botrytis cinerea Pers.:Fr., Fusarium oxysporum Schlechtend.:Fr., Sclerotium rolfsii Sacc., Rhizoctonia solani Kühn,, Botryosphaeria dothidea (Moug.:Fr.) Ces &; De Not. and Pythium ultimum Trow, using an agar diffusion method. Compared to the other plants as well as specific standard fungicides for each pathogen, the root extract of D. kilimandscharicus showed the highest broad-spectrum in vitro antifungal activity by inhibiting the mycelial growth of three of the six test organisms. Additionally, in vivo antifungal activity of dry powdered material from these plants against sorghum covered (Sporisorium sorghi) and loose kernel (Sporisorium cruentum) smuts was screened under field conditions by artificially inoculating separate sets of sorghum seed with smut spores before treating with plant material or two traditionally used fungicides, Thiram^® and Apron Plus^® that served as positive controls. Although the incidence of both loose and covered kernel smuts were significantly reduced by material from all plant species, the dry powdered berries of P. dodecandra were most effective. Compared to the untreated control, treatment with the plant material as well as standard fungicides resulted in significant yield increases. It was concluded that a rationale has been established for further investigation into the structured utilization of natural vegetation indigenous to Ethiopia in the agricultural industry.     

Micropropagation of Ajuga species: a mini review

The genus Ajuga L., belonging to Lamiaceae family, is widespread. The demand for Ajuga species has risen sharply because of their medicinal, ornamental, and pharmacological properties. These wide-ranging plants are being rapidly depleted due to over-collection for ornamental and medicinal purposes, as well as by habitat destruction and deforestation. Ajuga boninsimae, A. bracteosa, A. ciliate, A. genevensis, A. incisa, A. makinoi, A. multiflora, A. pyramidalis, A. shikotanensis, A. reptans, and A. vestita are categorized and protected as endangered plants. In vitro plant culture has therefore emerged for the conservation and mass clonal propagation of rare plants. This mini-review covers the current in vitro scenario in the propagation of Ajuga species. Adventitious or axillary shoots are initiated on the leaf, petiole and internodes, as well as roots, nodes, and shoot tip explants. Shoot induction is predominantly dependent on plant growth regulators added to the culture medium. Full- or half-strength Murashige and Skoog medium with or without auxin is used for in vitro rooting. Rooted shoots need to be acclimatized in the greenhouse with an estimated 82–100% survival rate.     

Rice inositol polyphosphate kinase gene (<Emphasis Type="Italic">OsIPK2</Emphasis>), a putative new player of gibberellic acid signaling,involves in modulation of shoot elongation and fertility

Gibberellic acid (GA) is an important plant hormone mediating plant growth and development throughout the life span. Although many GA biosynthesis genes and signaling components have been revealed, the signal transduction mechanisms from GA perception to physiological actions are still largely unclear. In this study, we investigated the functions of a rice (Oryza sativa) inositol polyphosphate kinase gene (OsIPK2) in rice growth and development, showing that OsIPK2 is a putative new player in GA signaling. OsIPK2 is widely expressed in rice with high accumulation in tender and rapidly dividing tissues. The OsIPK2 protein is mainly localized in the nucleus and plasma membrane. To study the biological roles of OsIPK2 in rice, RNA interference and overexpression transgenic plants were generated. OsIPK2 antisense plants exhibited taller seedling height and lower fertility rate than the wild type, while overexpression lines showed reduced plant height. Microarray and qRT-PCR assays showed that expression levels of several GA-related genes were altered in transgenic plants. Besides, down-regulation of OsIPK2 resulted in hypersensitivity to paclobutrazol (PAC), a GA biosynthesis inhibitor. We also described that the expression of OsIPK2 could be either induced by GA or repressed by PAC. Taken together, these findings suggested that OsIPK2 is likely a negative regulator of GA signaling and involves in modulating shoot elongation and fertility.