Mitigation of UV-B Radiation Stress in Tobacco Pollen by Expression of the Tardigrade Damage Suppressor Protein (Dsup).

Pollen, the male gametophyte of seed plants, is extremely sensitive to UV light, which may prevent fertilization. As a result, strategies to improve plant resistance to solar ultraviolet (UV) radiation are required. The tardigrade damage suppressor protein (Dsup) is a putative DNA-binding protein that enables tardigrades to tolerate harsh environmental conditions, including UV radiation, and was therefore considered as a candidate for reducing the effects of UV exposure on pollen. Tobacco pollen was genetically engineered to express Dsup and then exposed to UV-B radiation to determine the effectiveness of the protein in increasing pollen resistance. To establish the preventive role of Dsup against UV-B stress, we carried out extensive investigations into pollen viability, germination rate, pollen tube length, male germ unit position, callose plug development, marker protein content, and antioxidant capacity. The results indicated that UV-B stress has a significant negative impact on both pollen grain and pollen tube growth. However, Dsup expression increased the antioxidant levels and reversed some of the UV-B-induced changes to pollen, restoring the proper distance between the tip and the last callose plug formed, as well as pollen tube length, tubulin, and HSP70 levels. Therefore, the expression of heterologous Dsup in pollen may provide the plant male gametophyte with enhanced responses to UV-B stress and protection against harmful environmental radiation.

OMS alerta sobre impacto ambiental da indústria do tabaco

A Organização Mundial da Saúde (OMS) revelou nesta terça-feira (31) novas informações sobre o quanto o tabaco prejudica o meio ambiente e a saúde humana, pedindo medidas para tornar a indústria mais responsável pela destruição que está causando.

Regulation of Leaf Angle Protects Photosystem I under Fluctuating Light in Tobacco Young Leaves.

Fluctuating light is a typical light condition in nature and can cause selective photodamage to photosystem I (PSI). The sensitivity of PSI to fluctuating light is influenced by the amplitude of low/high light intensity. Tobacco mature leaves are tended to be horizontal to maximize the light absorption and photosynthesis, but young leaves are usually vertical to diminish the light absorption. Therefore, we tested the hypothesis that such regulation of the leaf angle in young leaves might protect PSI against photoinhibition under fluctuating light. We found that, upon a sudden increase in illumination, PSI was over-reduced in extreme young leaves but was oxidized in mature leaves. After fluctuating light treatment, such PSI over-reduction aggravated PSI photoinhibition in young leaves. Furthermore, the leaf angle was tightly correlated to the extent of PSI photoinhibition induced by fluctuating light. Therefore, vertical young leaves are more susceptible to PSI photoinhibition than horizontal mature leaves when exposed to the same fluctuating light. In young leaves, the vertical leaf angle decreased the light absorption and thus lowered the amplitude of low/high light intensity. Therefore, the regulation of the leaf angle was found for the first time as an important strategy used by young leaves to protect PSI against photoinhibition under fluctuating light. To our knowledge, we show here new insight into the photoprotection for PSI under fluctuating light in nature.

Genome-wide characterization, evolution, and expression profiling of FBA gene family in response to light treatments and abiotic stress in Nicotiana tabacum.

Fructose 1,6-bisphosphate aldolase (FBA) as a key enzyme play crucial roles in glycolysis, gluconeogenesis and Calvin cycle processes in plants. However, limited information is known regarding FBA genes in Nicotiana tabacum. In this study, 16 FBAs were identified and characterized in Nicotiana tabacum. Phylogenetic analysis revealed that these genes can be categorized as type I (NtFBA1-10 located in chloroplast) and type II (NtFBA11-16 located in cytoplasm) subfamilies. According to the conserved motifs and gene structure analysis, NtFBA protein sequences had the highly homologous to FBAs in other species. Most members of the NtFBA gene family responded positively to NaHCO3 stress, especially the expression of NtFBA13/14 increased by 642%. In addition, the expression results of NtFBAs under five abiotic stress (light, NaCl, NaHCO3, drought, and cold) conditions were showed that NtFBA13/14 were highly up-regulated. qRT-PCR results showed that most of the NtFBAs expressed higher in leaves. NtFBA7/8 and NtFBA13/14 have important significance in photosynthesis and abiotic stress, respectively. This study provides a basis foundation for further elucidating the function of NtFBAs and the N. tabacum mechanism of resistance under abiotic stress.

Red light imaging for programmed cell death visualization and quantification in plant-pathogen interactions.

Studies on plant-pathogen interactions often involve monitoring disease symptoms or responses of the host plant to pathogen-derived immunogenic patterns, either visually or by staining the plant tissue. Both these methods have limitations with respect to resolution, reproducibility, and the ability to quantify the results. In this study we show that red light detection by the red fluorescent protein (RFP) channel of a multipurpose fluorescence imaging system that is probably available in many laboratories can be used to visualize plant tissue undergoing cell death. Red light emission is the result of chlorophyll fluorescence on thylakoid membrane disassembly during the development of a programmed cell death process. The activation of programmed cell death can occur during either a hypersensitive response to a biotrophic pathogen or an apoptotic cell death triggered by a necrotrophic pathogen. Quantifying the intensity of the red light signal enables the magnitude of programmed cell death to be evaluated and provides a readout of the plant immune response in a faster, safer, and nondestructive manner when compared to previously developed chemical staining methodologies. This application can be implemented to screen for differences in symptom severity in plant-pathogen interactions, and to visualize and quantify in a more sensitive and objective manner the intensity of the plant response on perception of a given immunological pattern. We illustrate the utility and versatility of the method using diverse immunogenic patterns and pathogens.

Ultraviolet-B acclimation is supported by functionally heterogeneous phenolic peroxidases.

Tobacco plants were grown in plant chambers for four weeks, then exposed to one of the following treatments for 4 days: (1) daily supplementary UV-B radiation corresponding to 6.9 kJ m-2 d-1 biologically effective dose (UV-B), (2) daily irrigation with 0.1 mM hydrogen peroxide, or (3) a parallel application of the two treatments (UV-B + H2O2). Neither the H2O2 nor the UV-B treatments were found to be damaging to leaf photosynthesis. Both single factor treatments increased leaf H2O2 contents but had distinct effects on various H2O2 neutralising mechanisms. Non-enzymatic H2O2 antioxidant capacities were increased by direct H2O2 treatment only, but not by UV-B. In contrast, enzymatic H2O2 neutralisation was mostly increased by UV-B, the responses showing an interesting diversity. When class-III peroxidase (POD) activity was assayed using an artificial substrate (ABTS, 2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid)), both treatments appeared to have a positive effect. However, only UV-B-treated leaves showed higher POD activities when phenolic compounds naturally occurring in tobacco leaves (chlorogenic acid or quercetin) were used as substrates. These results demonstrate a substrate-dependent, functional heterogeneity in POD and further suggest that the selective activation of specific isoforms in UV-B acclimated leaves is not triggered by excess H2O2 in these leaves.

Expression of a Tardigrade Dsup Gene Enhances Genome Protection in Plants.

DNA damage is one of the most impactful events in living organisms, leading to DNA sequence changes (mutation) and disruption of biological processes. A study has identified a protein called Damage Suppressor Protein (Dsup) in the tardigrade Ramazzotius varieornatus that has shown to reduce the effects of radiation damage in human cell cultures (Hashimoto in Nature Communications 7:12808, 2016). We have generated tobacco plants that express the codon-optimized tardigrade Dsup gene and examined their responses when treated with mutagenic chemicals, ultraviolet (UV) and ionizing radiations. Our studies showed that compared to the control plants, the Dsup-expressing plants grew better in the medium containing mutagenic ethylmethane sulfonate (EMS). RT-qPCR detected distinct expression patterns of endogenous genes involved in DNA damage response and repair in the Dsup plants in response to EMS, bleomycin, UV-C and X-ray radiations. Comet assays revealed that the nuclei from the Dsup plants appeared more protected from UV and X-ray damages than the control plants. Overall, our studies demonstrated that Dsup gene expression enhanced tolerance of plants to genomutagenic stress. We suggest the feasibility of exploring genetic resources from extremotolerant species such as tardigrades to impart plants with tolerance to stressful environments for future climate changes and human space endeavors.

Photosynthetic characterization of flavodoxin-expressing tobacco plants reveals a high light acclimation-like phenotype.

Flavodoxins are electron carrier flavoproteins present in bacteria and photosynthetic microorganisms which duplicate the functional properties of iron-sulphur containing ferredoxins and replace them under adverse environmental situations that lead to ferredoxin decline. When expressed in plant chloroplasts, flavodoxin complemented ferredoxin deficiency and improved tolerance to multiple sources of biotic, abiotic and xenobiotic stress. Analysis of flavodoxin-expressing plants grown under normal conditions, in which the two carriers are present, revealed phenotypic effects unrelated to ferredoxin replacement. Flavodoxin thus provided a tool to alter the chloroplast redox poise in a customized way and to investigate its consequences on plant physiology and development. We describe herein the effects exerted by the flavoprotein on the function of the photosynthetic machinery. Pigment analysis revealed significant increases in chlorophyll a, carotenoids and chlorophyll a/b ratio in flavodoxin-expressing tobacco lines. Results suggest smaller antenna size in these plants, supported by lower relative contents of light-harvesting complex proteins. Chlorophyll a fluorescence and P700 spectroscopy measurements indicated that transgenic plants displayed higher quantum yields for both photosystems, a more oxidized plastoquinone pool under steady-state conditions and faster plastoquinone dark oxidation after a pulse of saturating light. Many of these effects resemble the phenotypes exhibited by leaves adapted to high irradiation, a most common environmental hardship faced by plants growing in the field. The results suggest that flavodoxin-expressing plants would be better prepared to cope with this adverse situation, and concur with earlier observations reporting that hundreds of stress-responsive genes were induced in the absence of stress in these lines.

Moderate heat stress accelerates photoinhibition of photosystem I under fluctuating light in tobacco young leaves.

Moderate heat stress and fluctuating light are typical conditions in summer in tropical and subtropical regions. This type of stress can cause photodamage to photosystems I and II (PSI and PSII). However, photosynthetic responses to the combination of heat and fluctuating light in young leaves are little known. In this study, we investigated chlorophyll fluorescence and P700 redox state under fluctuating light at 25 °C and 42 °C in young leaves of tobacco. Our results indicated that fluctuating light caused selective photodamage to PSI in the young leaves at 25 °C and 42 °C. Furthermore, the moderate heat stress significantly accelerated photoinhibition of PSI under fluctuating light. Within the first 10 s after transition from low to high light, cyclic electron flow (CEF) around PSI was highly stimulated at 25 °C but was slightly activated at 42 °C. Such depression of CEF activation at moderate heat stress were unable to maintain energy balance under high light. As a result, electron flow from PSI to NADP+ was restricted, leading to the over-reduction of PSI electron carriers. These results indicated that moderate heat stress altered the CEF performance under fluctuating light and thus accelerated PSI photoinhibition in tobacco young leaves.

Constitutive expression of a group 3 LEA protein from Medicago falcata (MfLEA3) increases cold and drought tolerance in transgenic tobacco.

KEY MESSAGE: MfLEA3 is involved in protection of catalase activity and confers multiple abiotic stress tolerance. Late embryogenesis abundant (LEA) proteins are involved in plant growth, development and abiotic stress tolerance. A member of group 3 LEA proteins from Medicago sativa subsp. falcata (L.) Arcang, MfLEA3, was investigated in the study. MfLEA3 transcript was induced in response to cold, dehydration, and abscisic acid (ABA), while the cold-induced transcript of MfLEA3 was blocked by pretreatment with inhibitor of ABA synthesis. Constitutive expression of MfLEA3 led to enhanced tolerance to cold, drought, and high-light stress in transgenic tobacco plants. Compared to accumulated reactive oxygen species (ROS) in the wild-type in response to treatments with low temperature, drought, and high light, ROS were not accumulated in transgenic plants. Superoxide dismutase, catalase (CAT), and ascorbate-peroxidase activities were increased in all plants after treatments with the above stresses, while higher CAT activity was maintained in transgenic plants compared with wild-type. However, transcript level of CAT-encoding genes including CAT1, CAT2, and CAT3 showed no significant difference between transgenic plants and wild-type, indicating that the higher CAT activity was not associated with its gene expression. ABA sensitivity and transcripts of several ABA and stress-responsive genes showed no difference between transgenic plant and wild-type, indicating that ABA signaling was not affected by constitutive expression of MfLEA3. The results suggest that MfLEA3 may be involved in the protection of CAT activity and confers multiple abiotic stress tolerance.

Responses of photosystem I compared with photosystem II to combination of heat stress and fluctuating light in tobacco leaves.

Moderate heat stress is usually accompanied with fluctuating light in summer. Although either heat stress or fluctuating light can cause photoinhibition of photosystems I and II (PSI and PSII), it is unclear whether moderate heat stress accelerate photoinhibition under fluctuating light. Here, we measured chlorophyll fluorescence, P700 redox state and the electrochromic shift signal under fluctuating light at 25 °C and 42 °C for tobacco leaves. We found that (1) the thylakoid proton conductance was significantly enhanced at 42 °C, leading to a decline in trans-thylakoid proton gradient (ΔpH); (2) this low ΔpH at 42 °C did not decrease donor-side limitation of PSI and thermal energy dissipation in PSII; (3) the activation of cyclic electron flow (CEF) around PSI was elevated at 42 °C; and (4) the moderate heat stress did not accelerate photoinhibition of PSI and PSII under fluctuating light. These results strongly indicate that under moderate heat stress the stimulation of CEF protects PSI under fluctuating light in tobacco leaves.

Red light delays programmed cell death in non-host interaction between Pseudomonas syringae pv tomato DC3000 and tobacco plants.

Light modulates almost every aspect of plant physiology, including plant-pathogen interactions. Among these, the hypersensitive response (HR) of plants to pathogens is characterized by a rapid and localized programmed cell death (PCD), which is critical to restrict the spread of pathogens from the infection site. The aim of this work was to study the role of light in the interaction between Pseudomonas syringae pv. tomato DC3000 (Pto DC3000) and non-host tobacco plants. To this end, we examined the HR under different light treatments (white and red light) by using a range of well-established markers of PCD. The alterations found at the cellular level included: i) loss of membrane integrity and nuclei, ii) RuBisCo and DNA degradation, and iii) changes in nuclease profiles and accumulation of cysteine proteinases. Our results suggest that red light plays a role during the HR of tobacco plants to Pto DC3000 infection, delaying the PCD process.

Limited Responsiveness of Chloroplast Gene Expression during Acclimation to High Light in Tobacco.

Acclimation to changing light intensities poses major challenges to plant metabolism and has been shown to involve regulatory adjustments in chloroplast gene expression. However, this regulation has not been examined at a plastid genome-wide level and for many genes, it is unknown whether their expression responds to altered light intensities. Here, we applied comparative ribosome profiling and transcriptomic experiments to analyze changes in chloroplast transcript accumulation and translation in leaves of tobacco (Nicotiana tabacum) seedlings after transfer from moderate light to physiological high light. Our time-course data revealed almost unaltered chloroplast transcript levels and only mild changes in ribosome occupancy during 2 d of high light exposure. Ribosome occupancy on the psbA mRNA (encoding the D1 reaction center protein of PSII) increased and that on the petG transcript decreased slightly after high light treatment. Transfer from moderate light to high light did not induce substantial alterations in ribosome pausing. Transfer experiments from low light to high light conditions resulted in strong PSII photoinhibition and revealed the distinct light-induced activation of psbA translation, which was further confirmed by reciprocal shift experiments. In low-light-to-high-light shift experiments, as well as reciprocal treatments, the expression of all other chloroplast genes remained virtually unaltered. Altogether, our data suggest that low light-acclimated plants upregulate the translation of a single chloroplast gene, psbA, during acclimation to high light. Our results indicate that psbA translation activation occurs already at moderate light intensities. Possible reasons for the otherwise mild effects of light intensity changes on gene expression in differentiated chloroplasts are discussed.

Plant Cell-Cell Transport via Plasmodesmata Is Regulated by Light and the Circadian Clock.

Plasmodesmata (PD) are essential for plant development, but little is known about their regulation. Several studies have linked PD transport to chloroplast-centered signaling networks, but the physiological significance of this connection remains unclear. Here, we show that PD transport is strongly regulated by light and the circadian clock. Light promotes PD transport during the day, but light is not sufficient to increase rates of PD transport at night, suggesting a circadian gating mechanism. Silencing expression of the core circadian clock gene, LHY/CCA1, allows light to strongly promote PD transport during subjective night, confirming that the canonical plant circadian clock controls the PD transport light response. We conclude that PD transport is dynamically regulated during the day/night cycle. Due to the many roles of PD in plant biology, this discovery has strong implications for plant development, physiology, and pathogenesis.

Pyrolysis, morphology and microwave absorption properties of tobacco stem materials.

The recent development of microwave radiation technology has increased the application possibilities of waste tobacco stems (WTSs). In this study, the morphology and microwave absorption properties of tobacco stem materials as well as the pyrolysis of the resultant biomass (BMTS) were studied via thermogravimetry-differential scanning calorimetry (TG-DSC), scanning electron microscopy (SEM), mercury intrusion porosimetry (MIP), and a vector network analysis (VNA). The results show that the BMTS pyrolysis involves four stages in air: dehydration, heat transfer, pyrolysis, and carbonisation, and it involves three stages in N2: moisture evaporation, de-volatilization, and charring. The microwave-assisted expansion of WTSs can improve the pore diameter and total porosity of the expanded tobacco stems (ETSs) and BMTS. The latter is a macroporous material with a total porosity of 78.2% and a probable pore size of 29.5 µm. Its pore size distribution ranges from 10.7 nm to 227 µm. The microwave absorption properties of the WTSs are affected by the moisture content, bulk density, and grain size; the properties can be enhanced by decreasing the grain size and increasing the moisture content and bulk density within the experimental range. The 3 dB bandwidth and amplitude vary by 0.45 MHz and - 0.406 dB per 1% increase in the moisture content of the materials, respectively. Our results demonstrate that tobacco stem materials with different moisture contents and grain sizes should be classified before the expansion or re-drying steps to ensure heating uniformity and product quality during the microwave radiation treatment.

Ambient conditions of elevated temperature and CO2 levels are detrimental to the probabilities of transmission by insects of a Potato virus Y isolate and to its simulated prevalence in the environment.

Conditions of elevated temperature and CO2 levels [30 °C and 970 parts-per-million (ppm), respectively] reduced the systemic titers of a potato virus Y (PVY) isolate in Nicotiana benthamiana plants, relative to standard conditions (25 °C, ~405 ppm CO2). Under controlled conditions we studied how these growing environments affected the transmission of infection by aphids. Probabilities of transmission of infection by insects that fed on infected donor plants kept at either standard conditions, or at 30 °C and 970 ppm CO2 were both determined and found to positively correlate with titers in donor leaves, independently of the ambient conditions in which recipient plantlets would grow. With these data, viral prevalence was simulated under conditions of elevated temperature and CO2 levels and found that for it to remain comparable to that simulated under standard conditions, insect arrivals to recipient plants in the former scenario would have to increase several-fold in their frequency.

UV-B strengthens antioxidant responses to drought in Nicotiana benthamiana leaves not only as supplementary irradiation but also as pre-treatment.

Potentials of UV-B (280-315 nm) radiation to alleviate effects of water deficit were studied using Nicotiana benthamiana plants in growth chambers. 10-days of limited watering resulted in 40% loss of soil water content as compared to well-watered controls. This drought was applied in three different ways: (i) in itself, (ii) after 4-days exposure of 6.9 kJ m-2 d-1 biologically effective supplementary UV-B radiation as pre-treatment, or (iii) in parallel with 6.9 kJ m-2 d-1 biologically effective supplementary UV-B. Responses were examined in two leaf groups: fully developed mature leaves (ML) and young leaves emerging during the 10-day treatment (YL). ML responded to UV-B or drought as single factor treatments with 7-14% loss of photochemical yield, while YL photochemistry was not decreased under the same conditions. The parallel two-factor treatment had no aggravating effect but alleviated drought-induced loss of leaf photochemistry in ML. Several positive single factor effects of drought or UV-B on antioxidants remained significant in the two-factor treatment both in ML and YL. Effects of the two factors applied in parallel were additive (equal to the sum of the effects caused by single factors separately) on total antioxidant capacities and singlet oxygen neutralizing; and synergistic (larger than the sum of single factor effects) on the flavonoid index in ML. A sequential application of UV-B and drought had additive positive effects on antioxidant capacity and flavonoid index of ML suggesting lasting effects of UV-B pre-treatment.

Signaling interactions between mitochondria and chloroplasts in Nicotiana tabacum leaf.

Research has begun to elucidate the signal transduction pathway(s) that control cellular responses to changes in mitochondrial status. Important tools in such studies are chemical inhibitors used to initiate mitochondrial dysfunction. This study compares the effect of different inhibitors and treatment conditions on the transcript amount of nuclear genes specifically responsive to mitochondrial dysfunction in leaf of Nicotiana tabacum L. cv. Petit Havana. The Complex III inhibitors antimycin A (AA) and myxothiazol (MYXO), and the Complex V inhibitor oligomycin (OLIGO), each increased the transcript amount of the mitochondrial dysfunction genes. Transcript responses to OLIGO were greater during treatment in the dark than in the light, and the dark treatment resulted in cell death. In the dark, transcript responses to AA and MYXO were similar to one another, despite MYXO leading to cell death. In the light, transcript responses to AA and MYXO diverged, despite cell viability remaining high with either inhibitor. This divergent response may be due to differential signaling from the chloroplast because only AA also inhibited cyclic electron transport, resulting in a strong acceptor-side limitation in photosystem I. In the light, chemical inhibition of chloroplast electron transport reduced transcript responses to AA, while having no effect on the response to MYXO, and increasing the response to OLIGO. Hence, when studying mitochondrial dysfunction signaling, different inhibitor and treatment combinations differentially affect linked processes (e.g. chloroplast function and cell fate) that then contribute to measured responses. Therefore, inhibitor and treatment conditions should be chosen to align with specific study goals.

Single-dose ß-aminobutyric acid treatment modifies tobacco (Nicotiana tabacum L.) leaf acclimation to consecutive UV-B treatment.

ß-Aminobutyric acid (BABA) pre-treatment has been shown to alter both biotic and abiotic stress responses. The present study extends this observation to acclimative UV-B-response, which has not been explored in this context so far. A single soil application of 300 ppm BABA modified the non-enzymatic antioxidant capacities and the leaf hydrogen peroxide levels in tobacco (Nicotiana tabacum L.) leaves in response to a 9-day treatment with 5.4 kJ m-2 d-1 biologically effective supplementary UV-B radiation in a model experiment that was performed in a growth chamber. BABA decreased leaf hydrogen peroxide levels both as a single factor and in combination with UV-B, but neither BABA nor UV-B affected leaf photochemistry significantly. The total antioxidant capacities were increased by either BABA or UV-B, and this response was additive in BABA pre-treated leaves. These results together with the observed changes in hydroxyl radical neutralising ability and non-enzymatic hydrogen peroxide antioxidant capacities show that BABA pre-treatment (i) has a long-term effect on leaf antioxidants even in the absence of other factors and (ii) modifies acclimative readjustment of prooxidant-antioxidant balance in response to UV-B. BABA-inducible antioxidants do not include phenolic compounds as a UV-B-induced increase in the adaxial leaf flavonoid index and total leaf extract UV absorption were unaffected by BABA.