Wheat Mesophyll Protoplasts Research Articles

A Leucine-Rich Repeat Receptor-like Kinase TaBIR1 Contributes to Wheat Resistance against Puccinia striiformis f. sp. tritici

Plant cell surface-localized receptor-like kinases (RLKs) recognize invading pathogens and transduce the immune signals inside host cells, subsequently triggering immune responses to fight off pathogen invasion. Nonetheless, our understanding of the role of RLKs in wheat resistance to the biotrophic fungus Puccinia striiformis f. sp. tritici (Pst) remains limited. During the differentially expressed genes in Pst infected wheat leaves, a Leucine-repeat receptor-like kinase (LRR-RLK) gene TaBIR1 was significantly upregulated in the incompatible wheat-Pst interaction. qRT-PCR verified that TaBIR1 is induced at the early infection stage of Pst. The transient expression of TaBIR1-GFP protein in N. bentamiana cells and wheat mesophyll protoplasts revealed its plasma membrane location. The knockdown of TaBIR1 expression by VIGS (virus induced gene silencing) declined wheat resistance to stripe rust, resulting in reduced reactive oxygen species (ROS) production, callose deposition, and transcripts of pathogenesis-related genes TaPR1 and TaPR2, along with increased Pst infection area. Ectopic overexpression of TaBIR1 in N. benthamiana triggered constitutive immune responses with significant cell death, callose accumulation, and ROS production. Moreover, TaBIR1 triggered immunity is dependent on NbBAK1, the silencing of which significantly attenuated the defense response triggered by TaBIR1. TaBIR1 interacted with the NbBAK1 homologues in wheat, co-receptor TaSERK2 and TaSERK5, the transient expression of which could restore the impaired defense due to NbBAK1 silencing. Taken together, TaBIR1 is a cell surface RLK that contributes to wheat stripe rust resistance, probably as a positive regulator of plant immunity in a BAK1-dependent manner.

Cell-penetrating peptide for targeted macromolecule delivery into plant chloroplasts.

Reports on chloroplast-targeted protein delivery using cell-penetrating peptides are scarce. In this study, a novel peptide-based macromolecule delivery strategy targeting chloroplasts was successfully developed in wheat mesophyll protoplasts. A peptide derived from the signal sequence of the chloroplast-targeted protein of ferredoxin-thioredoxin reductase catalytic chain of Spinacia oleracea with UniProtKB Id-P41348 exhibits properties of cellular internalization. DNase I was efficiently delivered into the chloroplast using 10 μM cTP with an efficiency of more than 90%. This cell-penetrating peptide-mediated approach offers various advantages over the existing chloroplast targeting methods, such as non-invasiveness, biocompatibility, low-toxicity, and target-specific delivery. The present study shows that peptide-based strategies hold tremendous potential in the field of chloroplast biotechnology. KEY POINTS: • Screening of database of chloroplast targeting peptides in order to develop an efficient cell-penetrating peptide termed as cTP. • cTP efficiently crosses the cell barrier and demonstrated chloroplast-localization. • cTP can be incorporated as a promising strategy for delivering macromolecules for crop improvement.

Autophagic degradation of the chloroplastic 2-phosphoglycolate phosphatase TaPGLP1 in wheat.

TaPGLP1, a chloroplast stromal 2-phosphoglycolate phosphatase of wheat, is an ATG8-interacting protein and undergoes autophagic degradation in starvation-treated wheat mesophyll protoplasts. Selective autophagy in plants has been shown to target diverse cellular cargoes including whole chloroplasts (Chlorophagy) and several chloroplast components (Piecemeal chlorophagy). Most cargoes of selective autophagy are captured by the autophagic machinery through their direct or indirect interactions with the autophagy-essential factor ATG8. Here, we reported a new ATG8-interacting cargo of piecemeal chlorophagy, the wheat photorespiratory 2-phosphoglycolate phosphatase TaPGLP1. The TaPGLP1-mCherry fusions expressed in wheat protoplasts located in the chloroplast stroma. Strikingly, these fusions are translocated into newly formed chloroplast surface protrusions after a long time incubation of protoplasts in a nutrition-free solution. Visualization of co-expressed TaPGLP1-mCherry and the autophagy marker GFP-TaATG8a revealed physical associations of TaPGLP1-mCherry-accumulating chloroplast protrusions with autophagic structures, implying the delivery of TaPGLP1-mCherry fusions from chloroplasts to the autophagic machinery. TaPGLP1-mCherry fusions were also detected in the GFP-TaATG8a-labelled autophagic bodies undergoing degradation in the vacuoles, which suggested the autophagic degradation of TaPGLP1. This autophagic degradation of TaPGLP1 was further demonstrated by the enhanced stability of TaPGLP1-mCherry in protoplasts with impaired autophagy. Expression of TaPGLP1-mCherry in protoplasts stimulated an enhanced autophagy level probably adopted by cells to degrade the over-produced TaPGLP1-mCherry fusions. Results from gene silencing assays showed the requirement of ATG2s and ATG7s in the autophagic degradation of TaPGLP1. Additionally, TaPGLP1 was shown to interact with ATG8 family members. Collectively, our data suggest that autophagy mediates the degradation of the chloroplast stromal protein TaPGLP1 in starvation-treated mesophyll protoplasts.

A Wheat WRKY Transcription Factor TaWRKY46 Enhances Tolerance to Osmotic Stress in transgenic Arabidopsis Plants.

WRKY transcription factors play central roles in developmental processes and stress responses of wheat. Most WRKY proteins of the same group (Group III) have a similar function in abiotic stress responses in plants. TaWRKY46, a member of Group III, was up-regulated by PEG treatment. TaWRKY46-GFP fusion proteins localize to the nucleus in wheat mesophyll protoplasts. Overexpression of TaWRKY46 enhanced osmotic stress tolerance in transgenic Arabidopsis thaliana plants, which was mainly demonstrated by transgenic Arabidopsis plants forming higher germination rate and longer root length on 1/2 Murashige and Skoog (MS) medium containing mannitol. Furthermore, the expression of several stress-related genes (P5CS1, RD29B, DREB2A, ABF3, CBF2, and CBF3) was significantly increased in TaWRKY46-overexpressing transgenic Arabidopsis plants after mannitol treatment. Taken together, these findings proposed that TaWRKY46 possesses vital functions in improving drought tolerance through ABA-dependent and ABA-independent pathways when plants are exposed to adverse osmotic conditions. TaWRKY46 can be taken as a candidate gene for transgenic breeding against osmotic stress in wheat. It can further complement and improve the information of the WRKY family members of Group III.

Monitoring autophagy in wheat living cells by visualization of fluorescence protein-tagged ATG8

Autophagy is a highly conserved eukaryotic degradation process during which bulk cytoplasmic materials are transported by double-membrane autophagosomes into the vacuole for degradation. Methods of monitoring autophagy are indispensable in studying the mechanism and functions of autophagy. AuTophaGy-related protein 8 (ATG8) functions in autophagosome assembly by decorating on autophagic membranes, and the inner membrane-bound ATG8 proteins enter the vacuole via active autophagy flux. Fluorescence protein (FP)-tagged forms of ATG8 have been explored as visual markers to monitor autophagy in animals and several plant species. Here, we evaluated and modified this FP-ATG8-based autophagy monitoring method in wheat (Triticum aestivum L.) by fluorescence observation of green fluorescence protein (GFP)-tagged and Discosoma red fluorescent protein (DsRED)-tagged forms of one wheat ATG8, TaATG8h, in wheat mesophyll protoplasts. Under a nutrient-starvation condition, punctate GFP/DsRED- TaATG8h fluorescence representing autophagosomes was clearly observed in the cytoplasm. The accumulation of GFP-TaATG8h-labeled autophagosomes was impaired by the autophagosome biogenesis inhibitor 3-methyladenine but enhanced by the vacuolar degradation inhibitor concanamycin A. In addition, accumulated spreading fluorescence was observed in the vacuole, indicating active autophagy fluxes which led to continuous degradation of GFP/DsRED-TaATG8h fusions and release of protease-tolerant free GFP/DsRED proteins in the vacuole. To observe FP-tagged TaATG8h in other types of wheat cell, we also expressed GFP-TaATG8h in leaf epidermal cells. Consistent with its performance in protoplasts, GFP-TaATG8h showed punctate fluorescence representing autophagosomes in leaf epidermal cells. Taken together, our results proved the feasibility of using FP-tagged ATG8 to monitor both autophagosome accumulation and autophagy flux in living wheat cells.

Overexpression of TaNAC2D Displays Opposite Responses to Abiotic Stresses between Seedling and Mature Stage of Transgenic Arabidopsis.

Environmental stresses frequently affect plant growth and development, and many genes have been found to be induced by unfavorable environmental conditions. Here, we reported the biological functions of TaNAC2D, a stress-related NAC (NAM, ATAF, and CUC) gene from wheat. TaNAC2D showed transcriptional activator activity in yeast. TaNAC2D-GFP fusion protein was localized in the nucleus of wheat mesophyll protoplasts. TaNAC2D transcript abundance was significantly induced by NaCl, PEG6000, and abscisic acid (ABA) at seedling stage, and repressed by NaCl and PEG6000 at mature plant stage. When TaNAC2D was introduced into Arabidopsis, the 35-day-old soil-grown TaNAC2D-overexpression (TaNAC2D-OX) plants displayed slower stomatal closure, higher water loss rate, and more sensitivity to salt and drought stresses compared with WT plants. In contrast, TaNAC2D-OX seedlings, grown on 1/2 MS medium supplemented with different concentrations of NaCl, Mannitol, and MV, had enhanced tolerances to salt, osmotic and oxidative stresses during seed germination and post-germination periods. The opposite stress-responsive phenotypes of transgenic Arabidopsis were consistent with the expression patterns of TaNAC2D in wheat. Moreover, under high salinity and dehydration conditions, three marker genes, including NCED3, RD29A, and RD29B, were down-regulated in 35-day-old TaNAC2D-OX plants grown in soil and up-regulated in 14-day-old TaNAC2D-OX seedlings grown on 1/2 MS medium. Our results suggest that the change in growth stages and environmental conditions may regulate TaNAC2D’s function.

Drought-responsive WRKY transcription factor genes TaWRKY1 and TaWRKY33 from wheat confer drought and/or heat resistance in Arabidopsis.

BackgroundDrought stress is one of the major causes of crop loss. WRKY transcription factors, as one of the largest transcription factor families, play important roles in regulation of many plant processes, including drought stress response. However, far less information is available on drought-responsive WRKY genes in wheat (Triticum aestivum L.), one of the three staple food crops.ResultsForty eight putative drought-induced WRKY genes were identified from a comparison between de novo transcriptome sequencing data of wheat without or with drought treatment. TaWRKY1 and TaWRKY33 from WRKY Groups III and II, respectively, were selected for further investigation. Subcellular localization assays revealed that TaWRKY1 and TaWRKY33 were localized in the nuclei in wheat mesophyll protoplasts. Various abiotic stress-related cis-acting elements were observed in the promoters of TaWRKY1 and TaWRKY33. Quantitative real-time PCR (qRT-PCR) analysis showed that TaWRKY1 was slightly up-regulated by high-temperature and abscisic acid (ABA), and down-regulated by low-temperature. TaWRKY33 was involved in high responses to high-temperature, low-temperature, ABA and jasmonic acid methylester (MeJA). Overexpression of TaWRKY1 and TaWRKY33 activated several stress-related downstream genes, increased germination rates, and promoted root growth in Arabidopsis under various stresses. TaWRKY33 transgenic Arabidopsis lines showed lower rates of water loss than TaWRKY1 transgenic Arabidopsis lines and wild type plants during dehydration. Most importantly, TaWRKY33 transgenic lines exhibited enhanced tolerance to heat stress.ConclusionsThe functional roles highlight the importance of WRKYs in stress response.Electronic supplementary materialThe online version of this article (doi:10.1186/s12870-016-0806-4) contains supplementary material, which is available to authorized users.

Association of VPg and eIF4E in the host tropism at the cellular level of Barley yellow mosaic virus and Wheat yellow mosaic virus in the genus Bymovirus

Barley yellow mosaic virus (BaYMV) and Wheat yellow mosaic virus (WYMV) are separate species in the genus Bymovirus with bipartite plus-sense RNA genomes. In fields, BaYMV infects only barley and WYMV infects only wheat. Here, we studied the replicative capability of the two viruses in barley and wheat mesophyll protoplasts. BaYMV replicated in both barley and wheat protoplasts, but WYMV replicated only in wheat protoplasts. The expression of wheat translation initiation factor 4E (eIF4E), a common host factor for potyviruses, from the WYMV genome enabled WYMV replication in barley protoplasts. Replacing the BaYMV VPg gene with that of WYMV abolished BaYMV replication in barley protoplasts, whereas the additional expression of wheat eIF4E from BaYMV genome restored the replication of the BaYMV mutant in barley protoplasts. These results indicate that both VPg and the host eIF4E are involved in the host tropism of BaYMV and WYMV at the replication level.

Apoplastic superoxide level in wheat protoplast under photooxidative stress is regulated by chloroplast redox signals: Effects on the antioxidant system

Apoplastic superoxide radical level in wheat mesophyll protoplasts was evaluated under different light intensities. Conditions that enhanced chloroplastic reactive oxygen species (ROS) production, such as high light intensity, paraquat treatments, or choloplastic Mn-superoxide dismutase (Mn-SOD) overexpression, increased the level of apoplastic superoxide. By contrast, the addition of diphenyl iodonium, a suicide NADPH oxidase inhibitor; DCMU, an herbicide that blocks plastoquinone reduction; and EGTA, a Ca 2+ chelator, decreased the superoxide level in the apoplast. Intracellular ROS production was rapidly stimulated by high light intensity and paraquat, and decreased in the presence of DCMU. The association between apoplastic superoxide level and total activities of the antioxidant enzymes superoxide dismutase (SOD) (E.C.:1.15.1.1), glutathione reductase (GR) (E.C.:1.6.4.2), ascobate peroxidase (APX) (E.C.:1.11.1.11), and catalase (CAT) (E.C.:1.11.1.6) was also evaluated. Changes in apoplastic superoxide level were positively correlated with activities of antioxidant enzymes mainly located in the chloroplasts, but not with CAT activity, which is located in peroxisomes and mitochondria. Regulation of apoplastic superoxide level by chloroplastic ROS and plastoquinone redox state is discussed, as well as its role in modulating antioxidant defense responses under photooxidative stress.

Mixing of maize and wheat genomic DNA by somatic hybridization in regenerated sterile maize plants.

Intergeneric somatic hybridization was performed between albino maize ( Zea mays L.) protoplasts and mesophyll protoplasts of wheat ( Triticum aestivum L.) by polyethylene glycol (PEG) treatments. None of the parental protoplasts were able to produce green plants without fusion. The maize cells regenerated only rudimentary albino plantlets of limited viability, and the wheat mesophyll protoplasts were unable to divide. PEG-mediated fusion treatments resulted in hybrid cells with mixed cytoplasm. Six months after fusion green embryogenic calli were selected as putative hybrids. The first-regenerates were discovered as aborted embryos. Regeneration of intact, green, maize-like plants needed 6 months of further subcultures on hormone-free medium. These plants were sterile, although had both male and female flowers. The cytological analysis of cells from callus tissues and root tips revealed 56 chromosomes, but intact wheat chromosomes were not observed. Using total DNA from hybrid plants, three RAPD primer combinations produced bands resembling the wheat profile. Genomic in situ hybridization (GISH) using total wheat DNA as a probe revealed the presence of wheat DNA islands in the maize chromosomal background. The increased viability and the restored green color were the most-significant new traits as compared to the original maize parent. Other intermediate morphological traits of plants with hybrid origin were not found.

Cell Cycle in Potentially Dedifferentiating Cereal Mesophyll Protoplasts Cultured in vitro II. Behaviour of the Cytoskeleton

Summary Following up on the recent finding that mesophyll protoplasts of wheat and oats were able to reenter the cell cycle when cultured in vitro , the behaviour of the cytoskeleton was studied. The aim was to elucidate whether changes in the cytoskeletal patterns correlate with the abnormal progression through the cycle observed previously. Protoplasts freshly isolated from mature mesophyll tissue (type B) exhibited very few, fragmented microtubules, whereas mesophyll protoplasts isolated from immature leaf tissue (type A) showed an abundant network fo microtubules. Type B protoplasts of wheat, which were able to reenter but recalcitrant to complete S-phase, failed to reestablish a microtubular network. Type A wheat protoplasts capable of progressing from G1 to G2 but incapable of entering mitosis lost microtubules during culturing and eventually resembled type B protoplasts. In contrast to wheat, type A protoplasts of oats retained microtubules in culture and type B protoplasts reestablished an abundant network of microtubules after about 5 days in culture. Both types of protoplasts were capable of going through mitosis. Microtubule configurations typical of mitotic cells were observed. However, several unusual features became apparent. No preprophase bands of microtubules were found. The spindles were short and blurred and did not form distinct poles. Phragmoplast microtubules were observed, but the deposition of cell plate material seemed to be slow. The phragmoplasts disappeared and a cortical network of microtubles was reestablished, apparently before completion of cytokinesis. Actin microfilaments were found in all freshly isolated and cultured premitotic protoplasts. They were not detected during the mitosis of oat protoplasts, but were found again in phragmoplasts. They appeared to be associated in abundance with presumptive phragmosomes, even after disappearance of phragmoplast microtubules. The cell cycle abnormalities observed indicate a possible cause for the cereal protoplast recalcitrance.

Subcellular compartmentation of pyrophosphate and alkaline pyrophosphatase in leaves

The subcellular localisation of pyrophosphate and alkaline pyrophosphatase in leaves has been studied using non-aqueous density gradient centrifugation of spinach leaves, and membrane filtration of wheat mesophyll protoplasts. The pyrophosphate was measured in extracts prepared in trichloroacetic acid, and could be quantitatively recovered from the leaf material. It was located predominantly in the cytosol, with a concentration of 0.2–0.3 mM. In contrast, the alkaline pyrophosphatase was largely, if not, exclusively, located in the chloroplast. By comparing the pyrophosphate levels in the cytosol with previously published data on the cytosolic levels of phosphate and metabolic intermediates, it is shown that the reactions catalysed by pyrophosphate: fructose-6-phosphate phosphotransferase and UDP-glucose pyrophosphorylase are close to the thermodynamic equilibrium and, thus, freely reversible in vivo. Comparison of the pyrophosphate levels with the reported electrical and pH gradient across the tonoplast membrane shows the free energy released during pyrophosphate hydrolysis is similar to that required to move a proton across the tonoplast membrane. It is suggested that pyrophosphate could operate as a secondary energy donor in the cytosol of plant cells.

Light-Induced Conversion of Nicotinamide Adenine Dinucleotide to Nicotinamide Adenine Dinucleotide Phosphate in Higher Plant Leaves

Light-induced conversion of NAD to NADP was investigated in higher plants. Upon illumination, conversion of NAD to NADP was observed in intact leaves of wheat and pea following incubation in the dark. This conversion was also observed in mesophyll protoplasts of wheat leaves when they were isolated in the dark or isolated in light and then preincubated in the dark. Chloroplasts isolated from wheat protoplasts prepared in the dark carried out the conversion. The conversion in the mechanically isolated spinach chloroplasts was observed only when they were isolated in the dark from leaves preincubated in darkness.Sucrose density gradient centrifugation of wheat protoplast extracts and differential centrifugation of protoplast extracts from various plants showed that most of the NAD kinase was localized in the chloroplasts. Therefore, the conversion of NAD to NADP is considered to occur in the chloroplasts. However, with extracts of maize mesophyll protoplasts, the enzyme was localized in the extrachloroplast fraction. The NAD kinase was activated some 30% by illumination of leaves or protoplasts of pea and wheat after preincubation in the dark.These results suggest that, in general, the light-induced conversion of NAD to NADP occurs in the chloroplast and is catalyzed by photoactivated NAD kinase using photochemically produced ATP.

Co-transport of Potassium and Sugars across the Plasmalemma of Mesophyll Protoplasts

Sugars (sucrose + hexoses) produced photosynthetically by isolated mesophyll protoplasts of wheat and tobacco were effluxed across the plasma membrane (3 to 10 micromoles hexose equivalents per milligram chlorophyll per hour). The efflux was sensitive to uncouplers and oligomycin which indicated a requirement for energy. A proton gradient was probably not coupled directly to the transport because changing the proton gradient across the plasma membrane by varying the pH of the medium or by adding sodium acetate had no significant effect on the rate of sugar release.A release of K(+) was associated with sugar efflux from the protoplasts. The molar ratio of K(+) to sugar varied between 1.5 and 2.5, depending on the species. Exogenous CKl, RbCl, and LiCl (50 millimolar each), but not NaCl or CsCl, significantly inhibited sugar efflux. Conditions that reduced sugar efflux (exogenous KCl, LiCl, mersalyl, or oligomycin) also reduced K(+) release and caused a time-dependent reduction in photosynthetic sucrose formation and increased amino acid and starch formation. Results obtained support the postulate that a K(+) symport is involved in the transport of sugar across the energized plasmalemma of photosynthetically active mesophyll cells.

Translocation

During photosynthesis by mesophyll protoplasts of wheat and tobacco, a linear efflux of sucrose and hexoses to the medium was observed, with the size of the intraprotoplast sugar pools remaining constant. Efflux of metabolites labeled by (14)CO(2) fixation was initially low because of dilution by internal pools, but increased exponentially with time. The results have significance both in terms of the mechanism of translocation and the use of isolated protoplasts in photosynthetic studies.

An Evaluation of Some Parameters Required for the Enzymatic Isolation of Cells and Protoplasts with CO2 Fixation Capacity from C3 and C4 Grasses

AbstractVariable factors affecting the enzymatic isolation of mesophyll protoplasts from Triticum aestivum (wheat), a C3 gras, and mesophyll protoplasts and bundle sheath strands from Digitaria sanguinalis (crabgrass), a C4 grass, have been examined with respect to yields and also photosynthetic capacity after isolation. Preparations with high yields and high photosynthetic capacity were obtained when small transverse leaf segments were incubated in enzyme medium in the light at 30°C, without mechanical shaking and without prior vacuum infiltration. Best results were obtained with an enzyme medium that included 0.5 M sorbitol, 1 mM MgCl2, 1 mM KH2PO4, 2% cellulase and 0.1% pectinase at pH 5.5. In gerneral, leaf age and leaf segment size were important factors, with highest yields and photosynthetic capacities obtained from young leaves cut into segments less than 0.8 mm. To facilitate the cutting of such small segments, a mechanical leaf cutter is described that uniformly (± 0.05 mm) cuts leaf tissue into transverse segments of variable size (0.4–2 mm). Isolations that required more than roughly 4 h gave poor yields with reduced photosynthetic capacity; however, using the optimum conditions described, functional preparations could be roughly 2 h. High rates of light dependent CO2 fixation by the C4 mesophyll protoplasts required the addition of pyruvate and low levels of oxalacetate, while isolated bundle sheath strands and C3 mesophyll protoplasts supported CO2 fixation without added substrates. Rates of CO2 fixation by isolated wheat protoplasts generally exceeded the reported rates of whole leaf photosynthesis. Wheat mesophyll protoplasts and crabgrass bundle sheath strands were stable when stored at 4°C while C4 mesophyll protoplasts were stable when stored at 25°C.