Mukund R. Shukla

Role of melatonin in alleviating cold stress in Arabidopsis thaliana

Melatonin (N‐acetyl‐5‐methoxytryptamine) has been implicated in abiotic and biotic stress tolerance in plants. However, information on the effects of melatonin in cold‐stress tolerance in vivo is limited. In this study, the effect of melatonin was investigated in the model plant Arabidopsis thaliana challenged with a cold stress at 4⁰C for 72 and 120 hr. Melatonin‐treated plants (10 and 30 μm) had significantly higher fresh weight, primary root length, and shoot height compared with the nontreated plants. To aid in the understanding of the role of melatonin in alleviating cold stress, we investigated the effects of melatonin treatment on the expression of cold‐related genes. Melatonin up‐regulated the expression of C‐repeat‐binding factors (CBFs)/Drought Response Element Binding factors (DREBs), a cold‐responsive gene, COR15a, a transcription factor involved in freezing and drought‐stress tolerance CAMTA1 and transcription activators of reactive oxygen species (ROS)‐related antioxidant genes, ZAT10 and ZAT12, following cold stress. The up‐regulation of cold signaling genes by melatonin may stimulate the biosynthesis of cold‐protecting compounds and contribute to the increased growth of plants treated with exogenous melatonin under cold stress.

Melatonin enhances the recovery of cryopreserved shoot tips of American elm (Ulmus americana L.).

Climate change and global migrations of people and goods have exposed trees to new diseases and abiotic challenges that threaten the survival of species. In vitro germplasm storage via cryopreservation is an effective tool to ensure conservation of tree species, but plant cells and tissues are exposed to multiple stresses during the cryopreservation process. The current study was designed to evaluate the potential of melatonin to improve survival through the process of cryopreservation. Shoot tips of in vitro‐grown plantlets and dormant winter buds of American elm were successfully cryopreserved in liquid nitrogen (LN) at −196°C under controlled environmental conditions following melatonin treatment and cold acclimation with either vitrification or encapsulation–vitrification protocols. Explants had optimal regrowth following cryopreservation when treated with the plant vitrification solution#2 (PVS2) for 10 min. Supplementation of both preculture and regrowth media with melatonin significantly enhanced regrowth of frozen shoots compared with the untreated control (P < 0.05). Approximately 80–100% of shoot explants grew under optimized conditions using melatonin‐enriched media. Shoot tips of dormant winter buds consistently produced nearly 100% regrowth with both techniques. The main steps of the optimized protocol are 14‐day cold‐acclimated cultures exposed to preculture medium with 0.1–0.5 μm melatonin for 24 hr, application of PVS2 for 10 min, rapid cooling in LN, rapid rewarming, removal of cryoprotectants, and recovery on a medium supplemented with 0.1–0.5 μm melatonin. Our results demonstrate the usefulness of the antioxidant melatonin for long‐term storage of naturally resistant elm germplasm.

Simultaneous induction of jasmonic acid and disease-responsive genes signifies tolerance of American elm to Dutch elm disease.

Dutch elm disease (DED), caused by three fungal species in the genus Ophiostoma, is the most devastating disease of both native European and North American elm trees. Although many tolerant cultivars have been identified and released, the tolerance mechanisms are not well understood and true resistance has not yet been achieved. Here we show that the expression of disease-responsive genes in reactions leading to tolerance or susceptibility is significantly differentiated within the first 144 hours post-inoculation (hpi). Analysis of the levels of endogenous plant defense molecules such as jasmonic acid (JA) and salicylic acid (SA) in tolerant and susceptible American elm saplings suggested SA and methyl-jasmonate as potential defense response elicitors, which was further confirmed by field observations. However, the tolerant phenotype can be best characterized by a concurrent induction of JA and disease-responsive genes at 96 hpi. Molecular investigations indicated that the expression of fungal genes (i.e. cerato ulmin) was also modulated by endogenous SA and JA and this response was unique among aggressive and non-aggressive fungal strains. The present study not only provides better understanding of tolerance mechanisms to DED, but also represents a first, verified template for examining simultaneous transcriptomic changes during American elm-fungus interactions.

Inhibition of phenylpropanoid biosynthesis increases cell wall digestibility, protoplast isolation, and facilitates sustained cell division in American elm (Ulmus americana)

BackgroundProtoplast technologies offer unique opportunities for fundamental research and to develop novel germplasm through somatic hybridization, organelle transfer, protoclonal variation, and direct insertion of DNA. Applying protoplast technologies to develop Dutch elm disease resistant American elms (Ulmus americana L.) was proposed over 30 years ago, but has not been achieved. A primary factor restricting protoplast technology to American elm is the resistance of the cell walls to enzymatic degradation and a long lag phase prior to cell wall re-synthesis and cell division.ResultsThis study suggests that resistance to enzymatic degradation in American elm was due to water soluble phenylpropanoids. Incubating tobacco (Nicotiana tabacum L.) leaf tissue, an easily digestible species, in aqueous elm extract inhibits cell wall digestion in a dose dependent manner. This can be mimicked by p-coumaric or ferulic acid, phenylpropanoids known to re-enforce cell walls. Culturing American elm tissue in the presence of 2-aminoindane-2-phosphonic acid (AIP; 10-150 μM), an inhibitor of phenylalanine ammonia lyase (PAL), reduced flavonoid content, decreased tissue browning, and increased isolation rates significantly from 11.8% (±3.27) in controls to 65.3% (±4.60). Protoplasts isolated from callus grown in 100 μM AIP developed cell walls by day 2, had a division rate of 28.5% (±3.59) by day 6, and proliferated into callus by day 14. Heterokaryons were successfully produced using electrofusion and fused protoplasts remained viable when embedded in agarose.ConclusionsThis study describes a novel approach of modifying phenylpropanoid biosynthesis to facilitate efficient protoplast isolation which has historically been problematic for American elm. This isolation system has facilitated recovery of viable protoplasts capable of rapid cell wall re-synthesis and sustained cell division to form callus. Further, isolated protoplasts survived electrofusion and viable heterokaryons were produced. Together, these results provide the first evidence of sustained cell division, callus regeneration, and potential application of somatic cell fusion in American elm, suggesting that this source of protoplasts may be ideal for genetic manipulation of this species. The technological advance made with American elm in this study has potential implications in other woody species for fundamental and applied research which require availability of viable protoplasts.

Melatonin and serotonin: Mediators in the symphony of plant morphogenesis

Melatonin and serotonin are important signaling and stress mitigating molecules that play important roles across growth and development in plants. Despite many well‐documented responses, a systematic investigation of the entire metabolic pathway (tryptophan, tryptamine, and N‐acetylserotonin) does not exist, leaving many open questions. The objective of this study was to determine the responses of Hypericum perforatum (L.) to melatonin, serotonin, and their metabolic precursors. Two well‐characterized germplasm lines (#4 and 112) created by mutation and a haploid breeding program were compared to wild type to identify specific responses. Germplasm line 4 has lower regenerative and photosynthetic capacity than either wild type or line 112, and there are documented significant differences in the chemistry and physiology of lines 4 and 112. Supplementation of the culture media with tryptophan, tryptamine, N‐acetylserotonin, serotonin, or melatonin partially reversed the regenerative recalcitrance and growth impairment of the germplasm lines. Quantification of phytohormones revealed crosstalk between the indoleamines and related phytohormones including cytokinin, salicylic acid, and abscisic acid. We hypothesize that melatonin and serotonin function in coordination with their metabolites in a cascade of phytochemical responses including multiple pathways and phytohormone networks to direct morphogenesis and protect photosynthesis in H. perforatum.

Plant Cryopreservation for Biotechnology and Breeding

Plant biodiversity is crucial for sustaining human life on our planet. More than 50,000 species are used globally for food, feed, fiber, medicine and horticulture. A wide range of plant-based biotechnological systems such as isolated root cultures, embryogenic cell and tissue cultures and cell suspensions are used in breeding programs, forestry and the production of pharmaceuticals. Cryopreservation is an essential tool for conservation and long-term maintenance of diverse germplasms with minimal requirements for cost and labor and a low risk of loss of preserved samples. However, large-scale use of cryogenic storage to back-up plant genetic collections is hampered by unavailability of effective methodology and genotype-specific responses of diverse specimens to cryoprotective treatments. Newly developed techniques such as droplet-vitrification are more effective and user-friendlier than classical methods of cryopreservation. Cryopreservation has been successfully employed for preserving several different types of plant materials. In this chapter we review various approaches to develop and improve cryopreservation protocols for diverse plant species. Applications of modern cryopreservation methods in biotechnology-based industry as well as breeding programs are also discussed.

Application of 3D printing to prototype and develop novel plant tissue culture systems

BackgroundDue to the complex process of designing and manufacturing new plant tissue culture vessels through conventional means there have been limited efforts to innovate improved designs. Further, development and availability of low cost, energy efficient LEDs of various spectra has made it a promising light source for plant growth in controlled environments. However, direct replacement of conventional lighting sources with LEDs does not address problems with uniformity, spectral control, or the challenges in conducting statistically valid experiments to assess the effects of light. Prototyping using 3D printing and LED based light sources could help overcome these limitations and lead to improved culture systems.ResultsA modular culture vessel design in which the fluence rate and spectrum of light are independently controlled was designed, prototyped using 3D printing, and evaluated for plant growth. This design is compatible with semi-solid and liquid based culture systems. Observations on morphology, chlorophyll content, and chlorophyll fluorescence based stress parameters from in vitro plants cultured under different light spectra with similar overall fluence rate indicated different responses in Nicotiana tabacum and Artemisia annua plantlets. This experiment validates the utility of 3D printing to design and test functional vessels and demonstrated that optimal light spectra for in vitro plant growth is species-specific.Conclusions3D printing was successfully used to prototype novel culture vessels with independently controlled variable fluence rate/spectra LED lighting. This system addresses several limitations associated with current lighting systems, providing more uniform lighting and allowing proper replication/randomization for experimental plant biology while increasing energy efficiency. A complete procedure including the design and prototyping of a culture vessel using 3D printing, commercial scale injection molding of the prototype, and conducting a properly replicated experiment are discussed. This open source design has the scope for further improvement and adaptation and demonstrates the power of 3D printing to improve the design of culture systems.

Establishment of invasive and non-invasive reporter systems to investigate American elm–Ophiostoma novo-ulmi interactions

Dutch elm disease (DED), caused by ascomycete fungi in the Ophiostoma genus, is the most devastating disease of American elm (Ulmus americana) trees. Cerato ulmin (CU), a hydrophobin secreted by the fungus, has been implicated in the development of DED, but its role in fungal pathogenicity and virulence remains uncertain and controversial. Here, we describe reporter systems based on the CU promoter and three reporter proteins (GFP, GUS and LUC), developed as research tools for quantitative and qualitative studies of DED in vitro, in vivo and in planta. A strain of the aggressive species Ophiostoma novo-ulmi was transformed with the reporter constructs using Agrobacterium-mediated transformation and the fungal transformants, namely M75-GFP, M75-GUS and M75-LUC, were examined for mitotic stability after repeated subcultures. The intensity of GFP fluorescence was strong in M75-GFP spores and hyphae, allowing microscopic investigations of spore structure, fungal morphogenesis and fungal development. The interaction of M75-GFP and U. americana callus cells was explored with scanning laser confocal microscopy facilitating qualitative studies on fungal strategies for the invasion and penetration of elm cells. M75-GUS was generated to provide an invasive, yet quantitative approach to study fungal-plant interactions in vitro and in planta. The generation of M75-LUC transformants was aimed at providing a non-destructive quantitative approach to study the role of CU in vivo. The sensitivity, low background signal and linearity of LUC assays all predict a very reliable approach to investigate and re-test previously claimed roles of this CU in fungal pathogenicity. These reporter systems provide new tools to investigate plant-pathogen interactions in this complex pathosystem and may aid in better understanding the development of DED.

In vitro propagation of cherry birch (Betula lenta L.)

Abstract: An efficient procedure for the micropropagation and conservation of cherry birch (Betula lenta L.), an endangered species in Canada, is reported. The model utilizes in vitro proliferation of fresh and dormant buds from both greenhouse and mature trees. Various factors were evaluated to optimize the protocol, including plant growth regulators, type and concentration of carbohydrates, and the composition of basal salts. 6-benzylaminopurine was the most effective cytokinin for shoot multiplication, with an optimal concentration of 5 µM. Sucrose was a more effective carbohydrate source than glucose, with the highest multiplication rate occurring at 3% sucrose (w/v). Shoot multiplication was similar on Murashige and Skoog (MS) and Driver and Kuniyuki Walnut (DKW) basal salts, but significantly lower on Woody Plant medium (WPM). Culture media with half-strength DKW basal salts and 20 µM indole-3-butyric acid was found to be the best for rooting (80%). Rooted plantlets were acclimatized to the greenhouse environment with a 37% survival rate. The micropropagation technology developed in the study offers excellent opportunities and tools for rapid replenishment of cherry birch trees in their natural environment, long-term conservation, and provides a platform for further research of this nationally endangered species.

Investigating the roles of phenylpropanoids in the growth and development of Zea mays L.

The Poaceae includes some of the most important food, fiber, and bio-fuel crops. While there have been many studies investigating the function of phenylpropanoids in this family, most of our understanding is based on correlative data rather than experimental evidence. The current study was conducted to evaluate the roles of phenylpropanoids in the growth and development of Zea mays and to develop an experimental model for further investigations. Z. mays seedlings were grown in vitro with various concentrations of the competitive phenylalanine ammonia lyase inhibitor, 2-aminoindane-2-phosphonic acid (AIP). Ferulic acid, a downstream biosynthetic product, was added to determine if it could rescue the induced phenotypes. At lower concentrations of AIP, plants exhibited elongated roots and shoots, but at higher concentrations, growth was extremely stunted. At the cellular level, the epidermal cells of roots cultured with AIP exhibited a loss of intercellular adhesion and organization, and their cell walls were more readily degraded by enzymatic digestion. These characteristics were accompanied by significant reductions in primary cell wall autofluorescence, indicating that less ferulic acid and other phenolics were incorporated in the cell wall. The majority of these symptoms could be partially or entirely rescued by ferulic acid, providing further evidence that these differences were due to the inhibition of phenylpropanoid biosynthesis. This study provides experimental evidence supporting and expanding upon hypothesized functions of phenylpropanoids in the growth and development of Z. mays and provides an experimental system for further investigations in the Poaceae and other taxonomic groups.