Leaf Protoplasts Research Articles

The Arabidopsis paraquat resistant1 mutant accumulates leucine upon dark treatment

The Arabidopsis paraquat resistant1 (PAR1) was classified as L-type amino acid transporter 4 (LAT4) based on a phylogenetic analysis of selected genes from Saccharomyces cerevisiae, Arabidopsis thaliana (L.) Heynh, and Homo sapiens that clustered LAT4 with four other members as a LAT family in Arabidopsis. In silico analysis of the Arabidopsis LATs identified an amino acid permease domain and motifs that are common in amino acid transporters. However, their role in amino acid transport remained to be studied. A knockout mutant for PAR1/LAT4 gene, reported here as par1-5, showed significantly altered growth compared with wild type on leucine-containing growth medium. Mutant par1-5 seedlings showed reduced biomass compared with wild type on nitrate-containing Murashige and Skoog growth medium, which was further reduced when grown on medium containing nitrate and leucine. Radio-labelled leucine uptake studies using leaf protoplasts and seedlings showed increased accumulation of leucine in par1-5 mutants compared with wild type. Increased accumulation of leucine in par1-5 was detected when seedlings or protoplasts were treated in the dark prior to isotopic feeding. These studies suggest that the PAR1/LAT4 protein, in addition to its ability to mediate paraquat and polyamine transport, possess leucine transport activity that is regulated by physiological conditions such as dark induction.

Functional Balancing of the Hypoxia Regulators RAP2.12 and HRA1 Takes Place in vivo in Arabidopsis thaliana Plants.

Plants are known to respond to variations in cellular oxygen availability and distribution by quickly adapting the transcription rate of a number of genes, generally associated to improved energy usage pathways, oxygen homeostasis and protection from harmful products of anaerobic metabolism. In terrestrial plants, such coordinated gene expression program is promoted by a conserved subfamily of ethylene responsive transcription factors called ERF-VII, which act as master activators of hypoxic gene transcription. Their abundance is directly regulated by oxygen through a mechanism of targeted proteolysis present under aerobic conditions, which is triggered by ERF-VII protein oxidation. Beside this, in Arabidopsis thaliana, the activity of the ERF-VII factor RAP2.12 has been shown to be restrained and made transient by the hypoxia-inducible transcription factor HRA1. This feedback mechanism has been proposed to modulate ERF-VII activity in the plant under fluctuating hypoxia, thereby enhancing the flexibility of the response. So far, functional balancing between RAP2.12 and HRA1 has been assessed in isolated leaf protoplasts, resulting in an inverse relationship between HRA1 amount and activation of RAP2.12 target promoters. In the present work, we showed that HRA1 is effective in balancing RAP2.12 activity in whole arabidopsis plants. Examination of a segregating population, generated from RAP2.12 and HRA1 over-expressing plants, led to the first quantitative proof that, over a range of either transgene expression levels, HRA1 counteracts the phenotypic and transcriptional effects of RAP2.12. This report supports the occurrence of fine-tuned regulation of the hypoxic response under physiological growth conditions.

Uptake, sequestration and tolerance of cadmium at cellular levels in the hyperaccumulator plant species Sedum alfredii.

Sedum alfredii is one of a few plant species known to hyperaccumulate cadmium (Cd). Uptake, localization, and tolerance of Cd at cellular levels in shoots were compared in hyperaccumulating (HE) and non-hyperaccumulating (NHE) ecotypes of Sedum alfredii. X-ray fluorescence images of Cd in stems and leaves showed only a slight Cd signal restricted within vascular bundles in the NHEs, while enhanced localization of Cd, with significant tissue- and age-dependent variations, was detected in HEs. In contrast to the vascular-enriched Cd in young stems, parenchyma cells in leaf mesophyll, stem pith and cortex tissues served as terminal storage sites for Cd sequestration in HEs. Kinetics of Cd transport into individual leaf protoplasts of the two ecotypes showed little difference in Cd accumulation. However, far more efficient storage of Cd in vacuoles was apparent in HEs. Subsequent analysis of cell viability and hydrogen peroxide levels suggested that HE protoplasts exhibited higher resistance to Cd than those of NHE protoplasts. These results suggest that efficient sequestration into vacuoles, as opposed to rapid transport into parenchyma cells, is a pivotal process in Cd accumulation and homeostasis in shoots of HE S. alfredii. This is in addition to its efficient root-to-shoot translocation of Cd.

Influence of culture vessels on mesophyll protoplast yield ofPelargoniumssp.

Somatic hybridization is a multistage process with many factors of influence. It requires a high yield of stable protoplasts. Plant protoplasts can be isolated from different plant tissues. Leaves of in vitro cultivated plants are one of the preferential sources. Using the same isolation protocol for four Pelargonium genotypes (Pelargonium ‘Pink Capitatum’, P. graveolens, P. × hortorum ‘Antik Violet’, wild type 585) the yield of leaf protoplasts from in vitro plants was very inconstant between the experiments, from high yield up to no protoplasts. To investigate the influence of culture conditions of the starting plants on the protoplast yield, plants were cultivated in Magenta vessels®, baby jars and 300 mL flasks. After 15 weeks, apex necrosis was observed in all vessels but with different strength: in Magenta® vessels it was lower, whereas plants cultivated in 300 mL flasks showed stronger symptoms. At the same time, these plants had a number of adventitious shoots with small leaves. Independently of the kind of culture vessel, ‘Pink Capitatum’ protoplasts could be isolated and they developed further in protoplast culture medium (PPM 1). The protoplast yield ranged between 1.6-5.0 × 105 protoplasts g-1 fresh weight (both from Magenta® vessel). Wild type 585 protoplasts were received also from all three plant culture conditions. However, getting protoplasts from Magenta® vessel plants involved some problems, the protoplast viability was low and a further development, at least a cell wall formation was not observed. Protoplasts of P. × hortorum could be isolated from all three variants. High number of protoplasts (1.8 × 106 g-1 fresh weight) was received from 300 mL flasks plants. Despite the high yield these protoplasts did not develop further. P. × hortorum protoplasts from Magenta® vessels showed cell divisions. A strong influence of culture vessels was found for P. graveolens protoplasts. Plants from 300 mL vessels gave stable protoplasts. These protoplasts showed a striking cell division activity. Plants from other vessels gave only once (from baby jar) viable protoplasts. The results show a considerable effect of plant culture conditions on Pelargonium protoplasts. Unfortunately, there is no vessel which would be optimal for all.

An active Mitochondrial Complex II Present in Mature Seeds Contains an Embryo-Specific Iron-Sulfur Subunit Regulated by ABA and bZIP53 and Is Involved in Germination and Seedling Establishment.

Complex II (succinate dehydrogenase) is an essential mitochondrial enzyme involved in both the tricarboxylic acid cycle and the respiratory chain. In Arabidopsis thaliana, its iron–sulfur subunit (SDH2) is encoded by three genes, one of them (SDH2.3) being specifically expressed during seed maturation in the embryo. Here we show that seed SDH2.3 expression is regulated by abscisic acid (ABA) and we define the promoter region (-114 to +49) possessing all the cis-elements necessary and sufficient for high expression in seeds. This region includes between -114 and -32 three ABRE (ABA-responsive) elements and one RY-enhancer like element, and we demonstrate that these elements, although necessary, are not sufficient for seed expression, our results supporting a role for the region encoding the 5’ untranslated region (+1 to +49). The SDH2.3 promoter is activated in leaf protoplasts by heterodimers between the basic leucine zipper transcription factors bZIP53 (group S1) and bZIP10 (group C) acting through the ABRE elements, and by the B3 domain transcription factor ABA insensitive 3 (ABI3). The in vivo role of bZIP53 is further supported by decreased SDH2.3 expression in a knockdown bzip53 mutant. By using the protein synthesis inhibitor cycloheximide and sdh2 mutants we have been able to conclusively show that complex II is already present in mature embryos before imbibition, and contains mainly SDH2.3 as iron–sulfur subunit. This complex plays a role during seed germination sensu-stricto since we have previously shown that seeds lacking SDH2.3 show retarded germination and now we demonstrate that low concentrations of thenoyltrifluoroacetone, a complex II inhibitor, also delay germination. Furthermore, complex II inhibitors completely block hypocotyl elongation in the dark and seedling establishment in the light, highlighting an essential role of complex II in the acquisition of photosynthetic competence and the transition from heterotrophy to autotrophy.

In vitro bioassay of allelopathy of Arabidopsis thaliana by sandwich method and protoplast co-culture method with digital image analysis.

We examined the allelopathic activities of Arabidopsis thaliana, ecotype Columbia by two in vitro methods. The effect of dried leaves on the growth of recipient lettuce seedlings was examined by the sandwich method. The allelopathic activity on protoplast growth was examined by co-culture with recipient lettuce leaf protoplasts in 50 µl liquid medium using a 96-well culture plate. Non-spherically enlarged or dividing protoplasts of lettuce were counted under an inverted microscope. Inhibition of yellow accumulation during lettuce protoplast growth was quantitated by image analysis of scanned digital images of 96-well culture plates. The results were described as the percentages of control without A. thaliana. The results were compared with those obtained using several plants which had strong allelopathic activities on recipient lettuce using the same methods. The growth of lettuce protoplasts was inhibited at both 4 and 8 days of culture with protoplasts of A. thaliana. The results suggested the usefulness of the protoplast co-culture method for future studies on allelopathy.

Transient Secretory Enzyme Expression in Leaf Protoplasts to Characterize SNARE Functional Classes in Conventional and Unconventional Secretion.

Despite a long case history, the use of protoplasts in cell biology research still divides scientists but their weaknesses can be exploited as strengths. Transient expression in protoplasts can saturate protein-protein interactions very efficiently, inhibiting the process of interest more efficiently than other approaches at gene expression level. The method described here consists of an assay providing a functional characterization of SNARE proteins in a heterogeneous population of cells, by the comparison of native and dominant negative mutant forms. In particular, it allows for discriminating between t-SNARE and i-SNARE functional classes.

Sound wave affects the expression of ethylene biosynthesis-related genes through control of transcription factors RIN and HB-1

We previously reported that sound wave treatment (1 kHz) delays fruit ripening in tomato (Solanum lycopersicum), affecting the expression of ethylene biosynthesis-related genes encoding 1-aminocyclopropane-1-carboxylic acid (ACC) synthases (ACS) and ACC oxidases (ACO). In this study, we investigated the activity of the transcription factors RIN and HB-1, which function in the ethylene biosynthetic pathway, in response to sound treatment. To investigate whether RIN and HB-1 directly activate the transcription of ACS and ACO, we performed transcriptional activation analysis in Arabidopsis thaliana leaf protoplasts, transiently expressing RIN or HB-1 and using reporter constructs with promoters of the tomato ACS and ACO genes. Activation of the endogenous AtACS and AtACO genes was also measured by qPCR. The RIN- and HB-1-induced expression of these genes decreased, but the HB-1-induced expression of some genes increased after sound treatment. To confirm these results, we performed transient assays in Nicotiana tabacum, which produced results similar to those observed in Arabidopsis. The major ethylene biosynthesis-related genes harbor a CArG-box as a RIN-binding motif. These findings indicate that RIN and HB-1 affect the expression of ethylene biosynthesis-related genes in response to sound treatment, and they suggest that RIN may regulate the ethylene biosynthesis-related genes by binding to their CArG-boxes.

Effects of root medium pH on root water transport and apoplastic pH in red-osier dogwood (Cornus sericea) and paper birch (Betula papyrifera) seedlings.

Soil pH is a major factor affecting plant growth. Plant responses to pH conditions widely vary between different species of plants. However, the exact mechanisms of high pH tolerance of plants are largely unknown. In the present study, we compared the pH responses of paper birch (Betula papyrifera) seedlings, a relatively sensitive species to high soil pH, with red-osier dogwood (Cornus sericea), reported to be relatively tolerant of high pH conditions. We examined the hypotheses that tolerance of plants to high root zone pH is linked to effective control of root apoplastic pH to facilitate nutrient and water transport processes In the study, we exposed paper birch and red-osier dogwood seedlings for six weeks to pH 5, 7 and 9 under controlled-environment conditions in hydroponic culture. Then, we measured biomass, gas exchange, root hydraulic conductivity, ferric chelate reductase (FCR) activity, xylem sap pH and the relative abundance of major elements in leaf protoplasts and apoplasts. The study sheds new light on the rarely studied high pH tolerance mechanisms in plants. We found that compared with paper birch, red-osier dogwood showed greater growth, higher gas exchange, and maintained higher root hydraulic conductivity as well as lower xylem sap pH under high pH conditions. The results suggest that the relatively high pH tolerance of dogwood is associated with greater water uptake ability and maintenance of low apoplastic pH. These traits may have a significant impact on the uptake of Fe and Mn by leaf cells.

Cuticular wax biosynthesis is positively regulated by WRINKLED4, an AP2/ERF‐type transcription factor, in Arabidopsis stems

The aerial surfaces of terrestrial plants are covered by a cuticular wax layer, which protects the plants from environmental stresses such as desiccation, high irradiance, and UV radiation. Cuticular wax deposition is regulated in an organ-specific manner; Arabidopsis stems have more than 10-fold higher wax loads than leaves. In this study, we found that WRINKLED4 (WRI4), encoding an AP2/ERF (ethylene-responsive factor) transcription factor (TF), is predominantly expressed in stem epidermis, is upregulated by salt stress, and is involved in activating cuticular wax biosynthesis in Arabidopsis stems. WRI4 harbors a transcriptional activation domain at its N-terminus, and fluorescent signals from a WRI4:eYFP construct were localized to the nuclei of tobacco leaf protoplasts. Deposition of epicuticular wax crystals on stems was reduced in wri4-1 and wri4-3 knockout mutants. Total wax loads were reduced by ~28% in wri4 stems but were not altered in wri4 siliques or leaves compared to the wild type. The levels of 29-carbon long alkanes, ketones, and secondary alcohols, which are the most abundant components of stem waxes, were significantly reduced in wri4 stems relative to the wild type. A transactivation assay in tobacco protoplasts and a chromatin immunoprecipitation (ChIP) assay showed that the expression of long-chain acyl-CoA synthetase1 (LACS1), β-ketoacyl CoA reductase1 (KCR1), PASTICCINO2 (PAS2), trans-2,3-enoyl-CoA reductase (ECR), and bifunctional wax synthase/acyl-CoA: diacylglycerol acyltransferase (WSD1) is positively regulated by direct binding of WRI4 to their promoters. Taken together, these results suggest that WRI4 is a transcriptional activator that specifically controls cuticular wax biosynthesis in Arabidopsis stems.

Viral RNase3 Co-Localizes and Interacts with the Antiviral Defense Protein SGS3 in Plant Cells.

Sweet potato chlorotic stunt virus (SPCSV; family Closteroviridae) encodes a Class 1 RNase III endoribonuclease (RNase3) that suppresses post-transcriptional RNA interference (RNAi) and eliminates antiviral defense in sweetpotato plants (Ipomoea batatas). For RNAi suppression, RNase3 cleaves double-stranded small interfering RNAs (ds-siRNA) and long dsRNA to fragments that are too short to be utilized in RNAi. However, RNase3 can suppress only RNAi induced by sense RNA. Sense-mediated RNAi involves host suppressor of gene silencing 3 (SGS3) and RNA–dependent RNA polymerase 6 (RDR6). In this study, subcellular localization and host interactions of RNase3 were studied in plant cells. RNase3 was found to interact with SGS3 of sweetpotato and Arabidopsis thaliana when expressed in leaves, and it localized to SGS3/RDR6 bodies in the cytoplasm of leaf cells and protoplasts. RNase3 was also detected in the nucleus. Co-expression of RNase3 and SGS3 in leaf tissue enhanced the suppression of RNAi, as compared with expression of RNase3 alone. These results suggest additional mechanisms needed for efficient RNase3-mediated suppression of RNAi and provide new information about the subcellular context and phase of the RNAi pathway in which RNase3 realizes RNAi suppression.

Possible mechanism involved with isolation protoplast of leaf

The process and mechanism of leaf protoplast formation after incubation to cell wall digesting solution was investigated. The acid material in the digesting solution provided the acid environment for leaves and led to the occurrence of acid degradation of cell wall, the separation between cell wall with protoplast and the escape of protoplast from cell wall into solution. The cytoplasm membrane inside the digesting solution was degraded by the acid environment and this accounted for the escape of cell nucleus and organelles from protoplast. In digesting solution, the recombination of organelles surrounding nuclear and subsequent formation of new protoplast with unanimous shape took place. These results showed that plant cell wall and cytoplasm membrane is the coat system of plant cell. The acid degradation of plant cell coat system makes recombination of cell organ and production of new protoplast possible.

Overexpression of Heat Shock Factor Gene HsfA3 Increases Galactinol Levels and Oxidative Stress Tolerance in Arabidopsis.

Heat shock factors (Hsfs) are central regulators of abiotic stress responses, especially heat stress responses, in plants. In the current study, we characterized the activity of the Hsf gene HsfA3 in Arabidopsis under oxidative stress conditions. HsfA3 transcription in seedlings was induced by reactive oxygen species (ROS), exogenous hydrogen peroxide (H2O2), and an endogenous H2O2 propagator, 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). HsfA3-overexpressing transgenic plants exhibited increased oxidative stress tolerance compared to untransformed wild-type plants (WT), as revealed by changes in fresh weight, chlorophyll fluorescence, and ion leakage under light conditions. The expression of several genes encoding galactinol synthase (GolS), a key enzyme in the biosynthesis of raffinose family oligosaccharides (RFOs), which function as antioxidants in plant cells, was induced in HsfA3 overexpressors. In addition, galactinol levels were higher in HsfA3 overexpressors than in WT under unstressed conditions. In transient transactivation assays using Arabidopsis leaf protoplasts, HsfA3 activated the transcription of a reporter gene driven by the GolS1 or GolS2 promoter. Electrophoretic mobility shift assays showed that GolS1 and GolS2 are directly regulated by HsfA3. Taken together, these findings provide evidence that GolS1 and GolS2 are directly regulated by HsfA3 and that GolS enzymes play an important role in improving oxidative stress tolerance by increasing galactinol biosynthesis in Arabidopsis.

A Putative Chloroplast Thylakoid Metalloprotease VIRESCENT3 Regulates Chloroplast Development in Arabidopsis thaliana

The chloroplast is the site of photosynthesis and many other essential plant metabolic processes, and chloroplast development is an integral part of plant growth and development. Mutants defective in chloroplast development can display various color phenotypes including the intriguing virescence phenotype, which shows yellow/white coloration at the leaf base and greening toward the leaf tip. Through large scale genetic screens, we identified a series of new virescent mutants including virescent3-1 (vir3-1), vir4-1, and vir5-1 in Arabidopsis thaliana. We showed that VIR3 encodes a putative chloroplast metalloprotease by map-based cloning. Through site-directed mutagenesis, we showed that the conserved histidine 235 residue in the zinc binding motif HEAGH of VIR3 is indispensable for VIR3 accumulation in the chloroplast. The chloroplast localization of VIR3 was confirmed by the transient expression of VIR3-GFP in leaf protoplasts. Furthermore, taking advantage of transgenic lines expressing VIR3-FLAG, we demonstrated that VIR3 is an intrinsic thylakoid membrane protein that mainly resides in the stromal lamellae. Moreover, topology analysis using transgenic lines expressing a dual epitope-tagged VIR3 indicated that both the N and C termini of VIR3 are located in the stroma, and the catalytic domain of VIR3 is probably facing the stroma. Blue native gel analysis indicated that VIR3 is likely present as a monomer or part of a small complex in the thylakoid membrane. This work not only implicates VIR3 as a new factor involved in early chloroplast development but also provides more insight into the roles of chloroplast proteases in chloroplast biogenesis.

Chemical Secretory Pathway Modulation in Plant Protoplasts.

The classical Golgi pathway is not the only mechanism for vacuolar protein transport in plants because alternative transport mechanisms have been described. The existence of these alternative pathways can be demonstrated using several chemicals and here we describe the use of brefeldin A (BFA), endo-β-N-acetylglucosaminidase H (Endo-H), and tunicamycin, on isolated tobacco leaf protoplasts. Two main methods are illustrated in this chapter, protoplast pulse-chase followed by protein immunoprecipitation, and protoplast immunofluorescence.

Silicate reduces cadmium uptake into cells of wheat

Cadmium (Cd) is a health threat all over the world and high Cd content in wheat causes high Cd intake. Silicon (Si) decreases cadmium content in wheat grains and shoot. This work investigates whether and how silicate (Si) influences cadmium (Cd) uptake at the cellular level in wheat. Wheat seedlings were grown in the presence or absence of Si with or without Cd. Cadmium, Si, and iron (Fe) accumulation in roots and shoots was analysed. Leaf protoplasts from plants grown without Cd were investigated for Cd uptake in the presence or absence of Si using the fluorescent dye, Leadmium Green AM. Roots and shoots of plants subjected to all four treatments were investigated regarding the expression of genes involved in the Cd uptake across the plasma membrane (i.e. LCT1) and efflux of Cd into apoplasm or vacuole from the cytosol (i.e. HMA2). In addition, phytochelatin (PC) content and PC gene (PCS1) expression were analysed. Expression of iron and metal transporter genes (IRT1 and NRAMP1) were also analysed. Results indicated that Si reduced Cd accumulation in plants, especially in shoot. Si reduced Cd transport into the cytoplasm when Si was added both directly during the uptake measurements and to the growth medium. Silicate downregulated LCT1 and HMA2 and upregulated PCS1. In addition, Si enhanced PC formation when Cd was present. The IRT1 gene, which was downregulated by Cd was upregulated by Si in root and shoot facilitating Fe transport in wheat. NRAMP1 was similarly expressed, though the effect was limited to roots. This work is the first to show how Si influences Cd uptake on the cellular level.

ZmbZIP91 regulates expression of starch synthesis-related genes by binding to ACTCAT elements in their promoters.

Starch synthesis is a key process that influences crop yield and quality, though little is known about the regulation of this complex metabolic pathway. Here, we present the identification of ZmbZIP91 as a candidate regulator of starch synthesis via co-expression analysis in maize (Zea mays L.). ZmbZIP91 was strongly associated with the expression of starch synthesis genes. Reverse tanscription-PCR (RT-PCR) and RNA in situ hybridization indicated that ZmbZIP91 is highly expressed in maize endosperm, with less expression in leaves. Particle bombardment-mediated transient expression in maize endosperm and leaf protoplasts demonstrated that ZmbZIP91 could positively regulate the expression of starch synthesis genes in both leaves and endosperm. Additionally, the Arabidopsis mutant vip1 carried a mutation in a gene (VIP1) that is homologous to ZmbZIP91, displayed altered growth with less starch in leaves, and ZmbZIP91 was able to complement this phenotype, resulting in normal starch synthesis. A yeast one-hybrid experiment and EMSAs showed that ZmbZIP91 could directly bind to ACTCAT elements in the promoters of starch synthesis genes (pAGPS1, pSSI, pSSIIIa, and pISA1). These results demonstrate that ZmbZIP91 acts as a core regulatory factor in starch synthesis by binding to ACTCAT elements in the promoters of starch synthesis genes.

Isolation and culture of grapevine protoplasts

Methods were established for isolation of protoplasts from different organs and tissues of grapevine plants grown in vitro . Cell division could not be induced in protoplasts from leaves, shoot tips and petioles of cv. Optima, whereas stein and root protoplasts showed division activity. Protoplasts derived from stems continued developing and formed microcalli and call. In experiments using stem protoplasts of several varieties, root and stem protoplasts divided in all cases; stem protoplasts of 4 varieties (Riesling, Kemer, Optima, Vidal) could be regenerated to callus. In leaf protoplasts, cell division could be induced only in case of cvs Vidal and Rupestris du Lot, however without formation of callus.

Molecular Characterization of TaFAR1 Involved in Primary Alcohol Biosynthesis of Cuticular Wax in Hexaploid Wheat.

Cuticular waxes are complex mixtures of very long chain (VLC) fatty acids and their derivatives in which primary alcohols are the most abundant components in the leaf surface of common wheat (Triticum aestivum L.). However, the genes involved in primary alcohol biosynthesis in wheat are still largely unknown. Here we identified, via a homology-based approach, the TaFAR1 gene belonging to the fatty acyl-CoA reductases (FARs) from wheat. Heterologous expression of TaFAR1 in yeast (Saccharomyces cerevisiae) and in the Arabidopsis (Arabidopsis thaliana) cer4-3 mutant afforded production of C22 primary alcohol and C22-C24 primary alcohols, respectively, and transgenic expression of TaFAR1 in tomato (Solanum lycopersicum) cv MicroTom leaves and fruits resulted in the accumulation of C26-C30 primary alcohols and C30-C34 primary alcohols, respectively. The TaFAR1 protein was localized to the endoplasmic reticulum (ER) in rice (Oryza sativa L.) leaf protoplasts. Moreover, the TaFAR1 expression pattern across various organs correlated with the levels of primary alcohols accumulating in corresponding waxes, and with the presence of platelet-shaped epicuticular wax crystals formed by primary alcohols. A nullisomic-tetrasomic wheat line lacking TaFAR1 had significantly reduced levels of primary alcohols in its leaf blade and anther wax. TaFAR1 was located on chromosome 4AL and appeared to be highly conserved, with only one haplotype among 32 wheat cultivars. Finally, TaFAR1 expression was induced by drought and cold stress in an ABA-dependent manner. Taken together, our results show that TaFAR1 is an active enzyme forming primary alcohols destined for the wheat cuticle.

Protoplast isolation and plant regeneration of guava (Psidium guajava L.) using experiments in mixture-amount design

A protocol was established for plant regeneration from leaf protoplasts of guava (Psidium guajava L.) using mixture-amount (concentration) experiments. A protoplast yield of 3.7 × 106 (viability >90 %) was obtained when 1 g leaf strips were digested in a solution of ∼0.75 M osmoticum with 6 % (w/v) enzyme containing cellulase: macerase: hemicellulase as proportion of ∼0.4: 0.5: 0.1. Protoplasts developed the maximum number of microcalli using 1.0 mg l−1 α-naphthaleneacetic acid (NAA). Maximum shoot formation (>12) via organogenesis from resulting calli was obtained using >3.4 mg l−1 PGRs containing kinetin (K): 6-benzylaminopurine (BAP) at a ratio of 0.6:0.4. Shoots were rooted in medium containing indole-3-butyric acid and plantlets were successfully acclimatized. Results of polynomial response models revealed that: (1) Osmolarity was the primary determinant for protoplast yield and viability, irrespective of osmoticum type; (2) Most of the variation in protoplast yield was driven by macerase concentration; (3) Protoplast viability was driven mainly by cellulase concentration; (4) NAA was superior to BAP for callus induction, an antagonistic proportional effect was observed when they were blended; and (5) K was more effective than BAP in shoot regeneration, but due to synergistic blending the response was highest when both were present. Overall, guava was amenable to protoplast culture and the mixture-amount design effectively characterized this protoplast system.