Model Plant Tobacco Research Articles

Activation of the antioxidant system and transduction of the mediated by exogenous calcium improve drought resistance in tobacco

Drought stress is one of the most important stresses in plants. The important role of calcium signal as a second messenger in alleviating drought stress is still unclear. We used tobacco as test material to simulate drought stress with 20 % PEG concentration. Physiological and transcriptome techniques were used to study how the model plant tobacco protects itself from drought stress by examining the mechanism used by exogenous calcium ions to respond to the antioxidant and hormone signals that indicate drought stress. Exogenous calcium was shown to increase the levels of expression of the proteins CDPK, CBL/CIPK and AQPs that respond to signaling by calcium under drought stress. The cis-acting elements of the promoters of the genes related to signals primarily focused on hormone and drought-related elements, which suggests that the exogenous calcium may mediate the transduction of hormone signals in response to drought stress. The results of physiological and transcriptional data showed that oxidative damage occurred in the tobacco leaves under drought stress. Under drought stress, exogenous calcium can enhance the activities of SOD (4.75 %), POD (42.33 %) and APX (512.43 %), up-regulate the expression of the genes that encode antioxidant enzymes to eliminate excessive reactive oxygen species (ROS), and alleviate the oxidative damage to tobacco leaves under drought stress. The calcium produced during drought stress increased the content of auxin (IAA), decreased that of abscisic acid (ABA) and downstream gene expression, and prevented excessive amplification of the ABA signal. Exogenous calcium enhanced the crosstalk between cytokinin, gibberellin and ABA signals.In summary, exogenous calcium can stimulate the synergistic effect of calcium and hormone signals and the antioxidant system, eliminate ROS, improve the crosstalk between plant hormones, and improve the drought resistance of tobacco.

Establishment of virus-induced gene-silencing system in Juglans sigillata Dode and functional analysis of JsFLS2 and JsFLS4

Juglans sigillata Dode is one of the important tree species in southwest China, and it has significant economic and ecological value. However, there is still a lack of effective methods to identify the functional genes of J. sigillata. By verifying the model plant tobacco, the pTRV2::JsPDS vector was able to cause photobleaching. This study showed that photobleaching occurred 24 and 30 d after the silencing vector was infected with aseptic seedlings and fruits of J. sigillata, respectively. When the OD600 was 0.6, and the injection dose was 500 μL, the gene silencing efficiency of aseptic seedlings was the highest at 16.7 %, significantly better than other treatments. Moreover, when the OD600 was 0.8, and the injection dose was 500 μL, the gene silencing efficiency in the walnut fruit was the highest (20 %). In addition, the VIGS system was successfully used to silence JsFLS2 and JsFLS4 genes in J. sigillata. This study also showed that the flavonol content and gene expression in the treatment group were decreased compared to the control group. In addition, the proteins transcribed and translated from the JsFLS4 gene may have higher catalytic activity for dihydroquercetin. The above results indicate that the TRV-mediated VIGS system can be an ideal tool for studying J. sigillata gene function.

Development of essential skills from a research project in biotechnology: coelomic fluid of earthworm induces adventitious roots in tobacco plants

Abstract- The goal of the study was to develop necessary skills for B. Tech students through an undergraduate project in biotechnology. Understanding of biotechnology involves keen observation of results and asking systematic and logical questions to find out the causes of key observation. This paper introduces teaching and attainment of essential skills during execution of their final year project. The project was focused on identifying the significance of coelomic fluid (CF) of earthworm in plant tissue culture medium by using the model plant tobacco. Project outcomes are: understanding the purpose of an experiment, conducting experiments in basic and applied areas of biotechnology, interpretation of results and communicate clearly during viva-voce examination and documenting the results in a scientific way. Essential skills viz: reading and understanding the literature, how to design an experiment with proper controls, logical explanation of results and communication skills were evaluated every month by rubrics designed by investigators. Assessment was further done by feedback system and surveys from the students. The project outcomes were matched with essential skills and their attainment was measured. Based on the evaluation it is concluded that this research project-based learning helped students to develop essential skills such as designing of experiments, logical explanation of results, communication and documentation of their observations with a conclusion in a scientific way. Keywords: coelomic fluid; earthworm; project-based skills; rubrics; tobacco

Unveiling the toxic effects, physiological responses and molecular mechanisms of tobacco (Nicotiana tabacum) in exposure to organic ultraviolet filters

Exposure to organic ultraviolet (UV) filters has raised concerns due to their potential adverse effects on environments. However, their toxic mechanisms on plants remain elusive. In this study, using integrative physiological and transcriptomic approaches we investigated the physiological and molecular responses to three representative UV filters, namely oxybenzone (OBZ), avobenzone (AVB), and octinoxate (OMC), in an agricultural model plant tobacco. The exposure to UV filters disrupts the functionality of photosystem reaction centers and the light-harvesting apparatus. Concurrently, UV filters exert a suppressive effect on the expression of genes encoding Rubisco and Calvin-Benson cycle enzymes, resulting in a decreased efficiency of the Calvin-Benson cycle and consequently hampering the process of photosynthesis. Exposure to UV filters leads to significant generation of reactive oxygen species within tobacco leaves and downregulation of oxidoreductase activities. Moreover, UV filters promote abscisic acid (ABA) accumulation by inducing the expression of ABA biosynthesis genes whereas repress indole-3-acetic acid (IAA) biosynthesis gene expression, which induce leaf yellowing and slow plant growth. In summary, the organic UV filters exert toxic effects on tobacco growth by inhibiting chlorophyll synthesis, photosynthesis, and the Calvin-Benson cycle, while generating excessive reactive oxygen species. This study sheds light on the toxic and tolerance mechanisms of UV filters in agricultural crops.

Panax notoginseng transcription factor WRKY15 modulates resistance to Fusarium solani by up-regulating osmotin-like protein expression and inducing JA/SA signaling pathways

BackgroundPanax notoginseng (Burk) F. H. Chen is a valuable traditional Chinese medicinal plant, but its commercial production is seriously affected by root rot caused by some pathogenic fungi, including Fusarium solani. Nevertheless, the genetic breeding for disease resistance of P. notoginseng remains limited. The WRKY transcription factors have been revealed to play important roles in plant defense responses, which might provide an inspiration for resistance improvement in P. notoginseng.ResultsIn this study, the regulatory mechanism of transcription factor PnWRKY15 on P. notoginseng resistance to F. solani infection was revealed. The suppressed expression of PnWRKY15 via RNA interference increased the sensitivity of P. notoginseng to F. solani and decreased the expression levels of some defense-related genes, including PnOLP1, which encodes an osmotin-like protein that confers resistance to F. solani. Ectopic expression of PnWRKY15 in the model plant tobacco significantly enhanced the resistance to F. solani. Moreover, the transcriptome sequencing analysis discovered that some pathogenesis-related genes were expressed at higher levels in the PnWRKY15-overexpressing tobacco than that in the wild-type tobacco. In addition, the jasmonic acid (JA) and salicylic acid (SA) signaling pathways were evidently induced by PnWRKY15-overexpression, that was evidenced by that the JA and SA contents were significantly higher in the PnWRKY15-overexpressing tobacco than that in the wild-type. Furthermore, PnWRKY15, which was localized in the nucleus, can trans-activate and up-regulate PnOLP1 expression according to the EMSA, yeast one-hybrid and co-expression assays.ConclusionsPnWRKY15 contributes to P. notoginseng resistance to F. solani by up-regulating the expression of resistance-related gene PnOLP1 and activating JA/SA signaling pathways. These findings will help to further elucidate the transcriptional regulatory mechanism associated with the P. notoginseng defense response to F. solani.

"Pathomorphogenic" Changes Caused by Citrus Bark Cracking Viroid and Transcription Factor TFIIIA-7ZF Variants Support Viroid Propagation in Tobacco.

Viroids are small, non-coding, pathogenic RNAs with the ability to disturb plant developmental processes. This dysregulation redirects the morphogenesis of plant organs, significantly impairing their functionality. Citrus bark cracking viroid (CBCVd) causes detrimental developmental distortions in infected hops (Humulus lupulus) and causes significant economic losses. CBCVd can infect cells and tissues of the model plant tobacco (Nicotiana tabacum), provided it is delivered via transgenesis. The levels of CBCVd in tobacco were enhanced in plant hybrids expressing CBCVd cDNAs and either the tobacco or hop variant of TFIIIA-7ZF, a viroid-mediated splicing derivative of transcription factor IIIA, which is important for viroid replication by DNA-dependent RNA polymerase II. The TFIIIA-7ZF variants can change the tobacco morphogenesis if expressed in leaves and shoots. In addition to the splitting of shoots, the "pathomorphogenic" network in hybrid plants expressing CBCVd and HlTFIIIA-7ZF induced leaf fusions and malformations. Moreover, CBCVd can dramatically change another morphogenesis into teratomic and petal-like tissues if propagated above some limit in young transgenic tobacco microspores and anthers. By comparative RNA profiling of transgenic tobacco shoots bearing TFIIIA-7ZFs and CBCVd-transformed/infected anthers, we found a differential expression of many genes at p < 0.05. As the main common factor showing the differential up-regulation in shoot and anther tissues, a LITTLE ZIPPER 2-like transcription factor was found. We propose that this factor, which can interact as a competitive inhibitor of the also dysregulated homeobox-leucin zipper family protein (HD-ZIPIII) in apical meristem, is essential for a network responsible for some morphological changes and modifications of plant degradome within shoot meristem regulation and secondary xylem differentiation.

Assessment of root-specific promoters in banana and tobacco and identification of a banana TIP2 promoter with strong root activity.

Genetic modification is one possible strategy to generate bananas (Musa spp.) with resistance to the soil-borne pathogen causing Fusarium wilt. The availability of banana root-specific promoters to target transgene expression to the sites of infection would be beneficial. We have assessed 18 promoter sequences derived from a range of plant species for their expression profiles in banana tissues to identify those with root-specific activity. Promoter sequences were isolated and fused to the β-glucuronidase (GUS) gene to assess their expression levels and tissue specificity in both banana and the model plant tobacco. Two heterologous promoters conferring high root expression levels in banana were identified, including a β‐glucosidase 1 (GLU1) promoter from maize and the RB7-type tonoplast intrinsic protein (TIP)-2 promoter from strawberry. Further, a novel Musa TIP2-2 promoter sequence was isolated and characterized which, when fused to the GUS gene, conferred very high GUS expression levels in banana roots. These promoters will expand the options for the control of gene expression in genetically modified bananas, providing a tool to develop plants with resistance not only to soil-borne diseases such as Fusarium wilt, but also for the improvement of other traits, such as nematode resistance, nutrition or abiotic stress resistance.

Introgression of SbERD4 Gene Encodes an Early-Responsive Dehydration-Stress Protein That Confers Tolerance against Different Types of Abiotic Stresses in Transgenic Tobacco.

Salicornia brachiata is an extreme halophyte that commonly grows on marsh conditions and is also considered a promising resource for drought and salt-responsive genes. To unveil a glimpse of stress endurance by plants, it is of the utmost importance to develop an understanding of stress tolerance mechanisms. ‘Early Responsive to Dehydration’ (ERD) genes are defined as a group of genes involved in stress tolerance and the development of plants. To increase this understanding, parallel to this expedited thought, a novel SbERD4 gene was cloned from S. brachiata, characterized, and functionally validated in the model plant tobacco. The study showed that SbERD4 is a plasma-membrane bound protein, and its overexpression in tobacco plants improved salinity and osmotic stress tolerance. Transgenic plants showed high relative water, chlorophylls, sugars, starch, polyphenols, proline, free amino acids, and low electrolyte leakage and H2O2 content compared to control plants (wild type and vector control) under different abiotic stress conditions. Furthermore, the transcript expression of antioxidant enzyme encoding genes NtCAT, NtSOD, NtGR, and NtAPX showed higher expression in transgenic compared to wild-type and vector controls under varying stress conditions. Overall, the overexpression of a novel early responsive to dehydration stress protein 4-encoding gene (SbERD4) enhanced the tolerance of the plant against multiple abiotic stresses. In conclusion, the overexpression of the SbERD4 gene mitigates plant physiology by enduring stress tolerance and might be considered as a promising key gene for engineering salinity and drought stress tolerance in crops.

Mutation of iPGAM using the CRISPR/Cas9 system affects pollen vitality and chlorophyll synthesis in Nicotiana tabacum

2, 3-bisphosphoglycerate-independent phosphoglycerate mutase (iPGAM), is a key enzyme that catalyzes the reversible interconversion of 3-phosphoglycerate and 2-phosphoglycerate in glycolysis. iPGAM has been found to be involved in several physiological processes, including stomatal movement, chlorophyll synthesis, pollen production, chloroplast development and photosynthesis in different plants. However, the roles iPGAM plays in model plant tobacco are still elusive. In the current study, two copies of the iPGAM gene in Nicotiana tabacum were cloned, NtiPGAMa and NtiPGAMb, from the variety “Honghuadajinyuan”. Expression profiles and subcellular localization analyses showed that NtiPGAMa and NtiPGAMb were highly expressed in the anthers and were localized to the nucleus, cytosol and cell membrane. Using the CRISPR/Cas9 system, three mutant lines with mosaic leaves were obtained by targeting the second exon of the iPGAM gene. Based on Sanger sequencing, the mutant lines were chimeric plants and displayed mutation rates ranging from 82.3% to 92.5%. The iPGAM mutations showed significantly decreased photosynthetic pigments, i.e., chlorophyll a, chlorophyll b and carotenoids, thinning or loss of chloroplast thylakoid lamellar structures. RT-PCR analysis showed that the expressions of genes associated with chlorophyll synthesis and photosynthesis were significantly down-regulated in the iPGAM mutant plants compared to wild type. The pollen development was greatly impaired in mutant plants, resulting in reduced and deformed pollens with grains inviable. Our findings indicate that iPGAM is critical for pollen vitality, chlorophyll synthesis and chloroplast morphogenesis in N. tabacum.

Novel PvACR3;2 and PvACR3;3 genes from arsenic-hyperaccumulator Pteris vittata and their roles in manipulating plant arsenic accumulation

Arsenite (AsIII) antiporter ACR3 is crucial for arsenic (As) translocation and sequestration in As-hyperaccumulator Pteris vittata, which has potential for phytoremediation of As-contaminated soils. In this study, two new ACR3 genes PvACR3;2 and PvACR3;3 were cloned from P. vittata and studied in model organism yeast (Saccharomyces cerevisiae) and model plant tobacco (Nicotiana tabacum). Both ACR3s mediated AsIII efflux in yeast, decreasing its As accumulation and enhancing its As tolerance. In addition, PvACR3;2 and PvACR3;3 were expressed in tobacco plant. Localized on the plasma membrane, PvACR3;2 mediated both AsIII translocation to the shoots and AsIII efflux from the roots in tobacco, resulting in 203 − 258% increase in shoot As after exposing to 5 μM AsIII under hydroponics. In comparison, localized to the vacuolar membrane, PvACR3;3 sequestrated AsIII in tobacco root vacuoles, leading to 18 − 20% higher As in the roots and 15 − 36% lower As in the shoots. Further, based on qRT-PCR, both genes were mainly expressed in P. vittata fronds, indicating PvACR3;2 and PvACR3;3 may play roles in AsIII translocation and sequestration in the fronds. This study provides not only new insights into the functions of new ACR3 genes in P. vittata, but also important gene resources for manipulating As accumulation in plants for phytoremediation and food safety.

Expressing Phosphate Transporter PvPht2;1 Enhances P Transport to the Chloroplasts and Increases Arsenic Tolerance in Arabidopsis thaliana.

Arsenic (As) contamination in soils is of great concerns due to its toxicity to plants. As an analogue, phosphorus plays an important role in protecting plants from As toxicity. In this study, we identified a new phosphate transporter 2 (PHT2), PvPht2;1, from As-hyperaccumulator Pteris vittata and analyzed its functions in As and P transport in a yeast mutant, and model plant Arabidopsis thalian. PvPht2;1 contained 12 transmembrane domains, sharing high identity with PHT2 genes in diverse plants. Further, independent of external P or As levels, PvPht2;1 was mainly expressed in P. vittata fronds with the expression being 3-4 folds higher than that in the roots and rhizomes. Localized to the chloroplasts based on GFP-fused PvPht2;1 in model plant tobacco, PvPht2;1 functioned as a low-affinity P transporter. Under As exposure, PvPht2;1 yeast transformants showed comparable growth with the control while high-affinity P transporter PvPht1;3 transformants showed better growth, suggesting that PvPht2;1 transported P but slower than PvPht1;3 transporter. Expressing PvPht2;1 in A. thaliana increased its shoot P concentration without influencing its As accumulation. Further, the chloroplasts' P content in transgenic A. thaliana increased by 37-59% than wild-type (WT) plants. Under As exposure, the photosynthesis of PvPht2;1-expressing A. thaliana remained stable but that of WT plants decreased. The data indicate that, under As stress, expressing PvPht2;1 in A. thaliana enhanced its P transport to the chloroplasts and protected its photosynthesis. In short, highly expressed in the fronds and not impacted by As exposure, chloroplast-located PvPht2;1 may have protected As-hyperaccumulator P. vittata from As toxicity by efficiently transporting only P to its chloroplasts.

Overexpression of Df4CL1 from Dryopteris fragrans Enhances Flavonoids and Lignin Production in Transgenic Tobacco

Dryopteris fragrans (L.) Schott is a fern in the family Dryopteridaceae that shows a good curative efficacy against several skin diseases, such as psoriasis and athlete’s foot. One of the substances that are effective is a flavonoid. The enzyme 4-Coumaric acid coenzyme A ligase (4CL) plays a key role in the phenylpropanoid metabolism pathway, which controls the metabolic direction of phenylpropanoids and is associated with the synthesis of flavonoids and lignin. In this paper, we examined the role of 4CL1 gene of D. fragrans (Df4CL1) by transferring it to the model plant tobacco. Then its gene function was analyzed through tissue expression, determination of protein and enzyme activity, and assessment of flavonoids and lignin production. The analysis showed that overexpression of Df4CL1 in the transgenic tobacco (TT) had higher protein content, 4CL activity, and content of flavonoids and lignin compared with wild type tobacco (WT). The tissue expression of Df4CL1 in TT was positively correlated with the content of flavonoids and lignin. Therefore, we conclude that Df4CL1 was associated with both flavonoids and lignin biosynthesis by controlling the relative abundance of flavonoids and lignin during normal development.

Panax notoginseng snakin gene increases resistance to Fusarium solani in transgenic tobacco

Snakins are cysteine-rich plant antimicrobial peptides that are important for plant defense responses to diverse pathogens, and a snakin gene, PnSN1, was cloned from Panax notoginseng to explore the role of snakin in the disease resistance mechanism of P. notoginseng. A gene expression analysis by quantitative real-time PCR revealed that PnSN1 expression was responsive to a Fusarium solani infection and was induced by four signaling molecules (methyl jasmonate, ethephon, salicylic acid, and hydrogen peroxide). A subcellular localization revealed that the PnSN1-GFP fusion protein was present only in the plant cell wall. Moreover, recombinant PnSN1 expressed in Escherichia coli was inhibitory toward the mycelial growth of four plant pathogenic fungi (F. solani, F. oxysporum, F. verticillioides, and Botryosphaeria dothidea), with increasing protein concentrations resulting in greater antifungal activities. The recombinant protein also inhibited F. solani spore germination. Additionally, the overexpression of PnSN1 in the model plant tobacco considerably increased the resistance to F. solani. Furthermore, the transient expression of the PnSN1 RNA interference construct increased the susceptibility of young P. notoginseng leaves to F. solani. These findings confirmed the importance of PnSN1 for the disease resistance of P. notoginseng.

Isolation of biotic stress resistance genes from cotton (Gossypium arboreum) and their analysis in model plant tobacco (Nicotiana tabacum) for resistance against cotton leaf curl disease complex

Cotton production is widely effected by Cotton Leaf Curl Virus (CLCuV) in world posing serious losses to cotton yield.The CRT genes from CLCuV resistant G. arboreum and CLCuV susceptible G. hirsutum were cloned and sequenced to know the differences of protein composition in both species. Molecular techniques were used to isolate full length putative biotic stress resistance genes from G. arboreum besides the analysis of identified novel genes in model plant tobacco (Nicotiana tabacum) for resistance to cotton leaf curl disease complex. It was found that transgenic plants over expressing Hydroperoxidelyase (HPL) genes exhibited higher enzyme activity than wild type. In addition the genome sequence information was used for the purpose of gene isolation. Even for the enhanced expression of Calreticulin (CRT), AOS and HPL in G. hirsutum, it still showed susceptibility against CLCuV suggesting alternative genes and pathways involved for the expression of resistance.

In silico characterization of SAMdC from Pokkali rice and its overexpression in transgenic tobacco

Polyamines help to mitigate salt stress in plants. SAMdC or S-adenosyl methionine decarboxylase is a crucial enzyme in the polyamine biosynthetic pathway. Overexpressing the samdc gene of Pokkali (a salt-tolerant Indica rice landrace) in the model plant tobacco through Agrobacterium-mediated transformation resulted in tobacco plants capable of withstanding high salinity (200 mM) stress. The transgenic tobacco plants maintained a steady level of the higher polyamines and resisted ionic imbalance under salt stress. Since appropriate binding of the substrate with the enzyme is essential for an enzymatic reaction, we performed molecular docking experiment of SAMdC enzyme of Pokkali rice and tobacco to get an idea about its commonality in the two plants in the backdrop of Japonica rice and Arabidopsis. In silico characterization of SAMdC revealed that the enzyme used the same substrate in Pokkali rice and tobacco, from where the gene is introgressed and where it is overexpressed.

Highly Resolved Systems Biology to Dissect the Etioplast-to-Chloroplast Transition in Tobacco Leaves.

Upon exposure to light, plant cells quickly acquire photosynthetic competence by converting pale etioplasts into green chloroplasts. This developmental transition involves the de novo biogenesis of the thylakoid system and requires reprogramming of metabolism and gene expression. Etioplast-to-chloroplast differentiation involves massive changes in plastid ultrastructure, but how these changes are connected to specific changes in physiology, metabolism, and expression of the plastid and nuclear genomes is poorly understood. Here, we describe a new experimental system in the dicotyledonous model plant tobacco (Nicotiana tabacum) that allows us to study the leaf deetiolation process at the systems level. We have determined the accumulation kinetics of photosynthetic complexes, pigments, lipids, and soluble metabolites and recorded the dynamic changes in plastid ultrastructure and in the nuclear and plastid transcriptomes. Our data describe the greening process at high temporal resolution, resolve distinct genetic and metabolic phases during deetiolation, and reveal numerous candidate genes that may be involved in light-induced chloroplast development and thylakoid biogenesis.

Up-regulation of lipid biosynthesis increases the oil content in leaves of Sorghum bicolor.

SummarySynthesis and accumulation of the storage lipid triacylglycerol in vegetative plant tissues has emerged as a promising strategy to meet the world's future need for vegetable oil. Sorghum (Sorghum bicolor) is a particularly attractive target crop given its high biomass, drought resistance and C4 photosynthesis. While oilseed‐like triacylglycerol levels have been engineered in the C3 model plant tobacco, progress in C4 monocot crops has been lagging behind. In this study, we report the accumulation of triacylglycerol in sorghum leaf tissues to levels between 3 and 8.4% on a dry weight basis depending on leaf and plant developmental stage. This was achieved by the combined overexpression of genes encoding the Zea mays WRI1 transcription factor, Umbelopsis ramanniana UrDGAT2a acyltransferase and Sesamum indicum Oleosin‐L oil body protein. Increased oil content was visible as lipid droplets, primarily in the leaf mesophyll cells. A comparison between a constitutive and mesophyll‐specific promoter driving WRI1 expression revealed distinct changes in the overall leaf lipidome as well as transitory starch and soluble sugar levels. Metabolome profiling uncovered changes in the abundance of various amino acids and dicarboxylic acids. The results presented here are a first step forward towards the development of sorghum as a dedicated biomass oil crop and provide a basis for further combinatorial metabolic engineering.

Overexpression of both AcSVP1 and AcSVP4 delays budbreak in kiwifruit A. chinensis var. deliciosa, but only AcSVP1 delays flowering in model plants

Kiwifruit SHORT VEGETATIVE PHASE (AcSVP) genes have been implicated in regulation of dormancy and flowering. Previously, we have shown that AcSVP2 and AcSVP3 have different effects on dormancy and flowering in transgenic kiwifruit and model plants. The role of two homologous genes, AcSVP1 and AcSVP4, remained unclear. Here, AcSVP1 and AcSVP4 were functionally characterized by ectopic expression in Actinidia species which show different chilling requirements for budbreak, as well as the model plant tobacco. Overexpression of AcSVP1 and AcSVP4 delayed spring budbreak in a high-chill A. chinensis var. deliciosa, but did not affect budbreak and flowering in a low-chill A. eriantha. Overexpression of AcSVP1 delayed flowering and seed germination in tobacco, while overexpression of AcSVP4 had only minor effects. Analysis of cis-regulatory motifs in the promoter regions of AcSVP1 and AcSVP4 and promoter transactivation assays revealed that AcSVP1 and AcSVP4 may be controlled by similar transcription factors, suggesting a conserved regulatory mechanism and a synergistic role for AcSVP1 and AcSVP4 in preventing premature kiwifruit budbreak during winter.

Apple Proliferation Phytoplasma Influences the Pattern of Plant Volatiles Emitted Depending on Pathogen Virulence

Apple proliferation (AP) and pear decline (PD) are the most severe diseases in pome fruit growing areas. AP-infected trees show typical symptoms such as witches’ broom, enlarged stipules, tasteless and dwarf fruits. PD-infected pears show a progressive weakening, reduced terminal growth, smaller fruits and die within weeks (quick decline) or years (slow decline). The diseases are caused by the cell-wall lacking bacteria Candidatus Phytoplasma mali (AP phytoplasma) and Ca. P. pyri (PD phytoplasma), respectively. In previous studies it has been shown that AP-infected apple trees emitted higher amounts of the sesquiterpene β-caryophyllene, an attractant of the insect vector Cacopsylla picta (Hemiptera: Psyllidae), thereby facilitating the dispersal of AP phytoplasma. In the present study, volatile organic compounds (VOCs) occurring in the headspace of plants infected with Ca. P. mali strains causing different severity of symptoms in apple plants were collected, analyzed and identified. Headspace samples from healthy and AP-infected model plant tobacco (Nicotiana occidentalis) and apple (Malus domestica) as well as from healthy and PD-infected pear (Pyrus communis) were investigated via thermodesorption and GC-MS analysis. Significantly higher concentrations of ethyl benzoate were produced in all phytoplasma-infected plants compared to healthy ones and an as yet unidentified sesquiterpene differed between the odor bouquets of healthy and by Ca. P. mali infected tobacco plants. Additionally, statistically significant higher amounts of both compounds were measured in the headspace of plants infected by the virulent AP strain. In apple, significantly higher concentrations of ethyl benzoate and methyl salicylate were observed for trees infected with strains of Ca. P. mali. Ethyl benzoate was also detected in the headspace of pear trees infected with Ca. P. pyri.

Improving recombinant Rubisco biogenesis, plant photosynthesis and growth by coexpressing its ancillary RAF1 chaperone

Enabling improvements to crop yield and resource use by enhancing the catalysis of the photosynthetic CO2-fixing enzyme Rubisco has been a longstanding challenge. Efforts toward realization of this goal have been greatly assisted by advances in understanding the complexities of Rubisco's biogenesis in plastids and the development of tailored chloroplast transformation tools. Here we generate transplastomic tobacco genotypes expressing Arabidopsis Rubisco large subunits (AtL), both on their own (producing tob(AtL) plants) and with a cognate Rubisco accumulation factor 1 (AtRAF1) chaperone (producing tob(AtL-R1) plants) that has undergone parallel functional coevolution with AtL. We show AtRAF1 assembles as a dimer and is produced in tob(AtL-R1) and Arabidopsis leaves at 10-15 nmol AtRAF1 monomers per square meter. Consistent with a postchaperonin large (L)-subunit assembly role, the AtRAF1 facilitated two to threefold improvements in the amount and biogenesis rate of hybrid L8(A)S8(t) Rubisco [comprising AtL and tobacco small (S) subunits] in tob(AtL-R1) leaves compared with tob(AtL), despite >threefold lower steady-state Rubisco mRNA levels in tob(AtL-R1). Accompanying twofold increases in photosynthetic CO2-assimilation rate and plant growth were measured for tob(AtL-R1) lines. These findings highlight the importance of ancillary protein complementarity during Rubisco biogenesis in plastids, the possible constraints this has imposed on Rubisco adaptive evolution, and the likely need for such interaction specificity to be considered when optimizing recombinant Rubisco bioengineering in plants.