Starch Staining Research Articles

Decoding Huanglongbing: Understanding the Origins, Impacts, and Remedies for Citrus Greening

Citrus cultivation is a vital component of the agricultural sector, offering significant nutritional and economic benefits. However, citrus plants face numerous disorders, including Huanglongbing (HLB), also known as citrus greening disease. HLB is a devastating disease caused by the bacterial species Candidatus Liberibacter asiaticus (Las), primarily transmitted by the citrus psyllid and grafting. Investigating the distribution and molecular characterization of HLB in various citrus varieties and psyllid populations is crucial. Molecular techniques and iodine starch staining have been used to detect HLB symptoms. Several studies have been designed to determine the incidence and severity of HLB symptoms in different citrus varieties and locations and estimate the psyllid population density in different citrus varieties. Furthermore, these studies established a correlation between the population of psyllids and the incidence and severity of HLB in different locations and these findings suggest a potential link between psyllid infestation and the prevalence of HLB. This study highlights the prevalence of HLB in various locations and varieties, providing insights into the distribution, severity, and correlation between psyllid populations and HLB incidence. The reliability of PCR testing for Las infection confirms its effectiveness. Such findings suggest implementing integrated pest management strategies, including monitoring and controlling psyllid populations, removing infected plants, and using certified disease-free planting materials to prevent HLB spread.

A rapid method to quantify vein density in C4 plants using starch staining.

C4 photosynthesis has evolved multiple times in the angiosperms and typically involves alterations to the biochemistry, cell biology and development of leaves. One common modification found in C4 plants compared with the ancestral C3 state is an increase in vein density such that the leaf contains a larger proportion of bundle sheath cells. Recent findings indicate that there may be significant intraspecific variation in traits such as vein density in C4 plants but to use such natural variation for trait-mapping, rapid phenotyping would be required. Here we report a high-throughput method to quantify vein density that leverages the bundle sheath-specific accumulation of starch found in C4 species. Starch staining allowed high-contrast images to be acquired permitting image analysis with MATLAB- and Python-based programmes. The method works for dicotyledons and monocotolydons. We applied this method to Gynandropsis gynandra where significant variation in vein density was detected between natural accessions, and Zea mays where no variation was apparent in the genotypically diverse lines assessed. We anticipate this approach will be useful to map genes controlling vein density in C4 species demonstrating natural variation for this trait.

Establishment of evaluation criterion based on starch dyeing method and implementation of optical and acoustic techniques for postharvest determination of “HongYang” kiwifruit ripeness

Realizing online detection of ripeness is meaningful and profitable in existing postharvest sorting system. This paper aims to establish evaluation criterion of kiwifruit ripeness based on starch dyeing method and realize online detection of kiwifruit ripeness based on spectroscopy and acoustic vibration. Considering storage time, flavor, and edible status of kiwifruit, evaluation criterion of kiwifruit ripeness was established based on starch conversion rate obtained from starch staining images. Differences were found in quality attributes (i.e., SSC and firmness), microstructure, absorbance spectra, and frequency-domain signals of kiwifruit at different ripeness levels. Based on spectral and acoustic vibration data collected by self-designed online systems, ripeness determination models were established. Linear PLSDA model performed better in spectral classification, while nonlinear SVM model performed better in acoustic vibration classification. Deep 1D-CNN models were proposed to further improve performance of ripeness determination models. Further novelty of this work was that it externally tested robustness of ripeness determination models using independent sample set with biological variability. For new batch of samples, overall accuracy of ripeness determination model based on spectral data (Accuracy = 93.08%) was slightly better than that of ripeness determination model based on acoustic vibration data (Accuracy = 92.31%). External test results showed that recognition performance of deep 1D-CNN model based on spectral data and deep 1D-CNN model based on acoustic vibration data was satisfactory. Deep 1D-CNN models proposed in this study can be used for determination of kiwifruit at different ripeness levels and have strong practicability in commercial online sorting.

The transcription factor AtHB23 modulates starch turnover for root development and plant survival under salinity

AtHB23 is a homeodomain-leucine zipper I transcription factor, previously characterized as a modulator of lateral root initiation and higher-order roots development. The role of this gene in response to salinity stress was largely unknown. To elucidate the role of AtHB23 in response to salinity stress, we combined histochemical β-glucuronidase (GUS) analysis, root phenotyping, starch staining, optic and transmission electron microscopy, expression studies by RT-qPCR, and transcriptome analysis of silenced, overexpressor, and crossed plants. We revealed that the expression pattern of AtHB23 is regulated by NaCl in the primary and lateral roots, affecting the root phenotype. A severe reduction in primary root length, a significant increment in the initiation of lateral roots, and a low survival rate in salinity conditions were observed in AtHB23 -silenced plants, whereas AtHB23 overexpressors showed the opposite phenotype. These developmental defects were explained by the degradation of starch granules and an alteration in starch metabolism. The AtHB23-target gene LAX3 is repressed in the tip of the primary root and affected by NaCl. We conclude that the lack of AtHB23 severely compromises plant survival and adaptation to salt stress conditions because this gene mediates starch granule turnover. • The transcription factor AtHB23 plays a crucial role under salinity stress. • AtHB23 silenced roots are unable to grow in NaCl. • Starch granules in amiR23 silenced plants are fully degraded. • The AtHB23 target, LAX3, is repressed by salinity in the root tip.

Mutation of the OsSAC1 Gene, which Encodes an Endoplasmic Reticulum Protein with an Unknown Function, Causes Sugar Accumulation in Rice Leaves.

Sugars are the most abundant organic compounds produced by plants, and can be used to build carbon skeletons and generate energy. The sugar accumulation 1 (OsSAC1) gene encodes a protein with an unknown function that exhibits four N-terminal transmembrane regions and two conserved domains of unknown function, DUF4220 and DUF594. OsSAC1 was found to be poorly and specifically expressed at the bottoms of young leaves and in the developing leaf sheaths. Subcellular location results showed that OsSAC1 was co-localized with ER:mCherry and targeted the endoplasmic reticulum (ER). OsSAC1 has been found to affect sugar partitioning in rice (Oryza sativa). I2/KI starch staining, ultrastructure observations and starch content measurements indicated that more and larger starch granules accumulated in ossac1 source leaves than in wild-type (WT) source leaves. Additionally, higher sucrose and glucose concentrations accumulated in the ossac1 source leaves than in WT source leaves, whereas lower sucrose and glucose concentrations were observed in the ossac1 young leaves and developing leaf sheaths than in those of the WT. Much greater expression of OsAGPL1 and OsAGPS1 (responsible for starch synthesis) and significantly less expression of OscFBP1, OscFBP2, OsSPS1 and OsSPS11 (responsible for sucrose synthesis) and OsSWEET11, OsSWEET14 and OsSUT1 (responsible for sucrose loading) occurred in ossac1 source leaves than in WT source leaves. A greater amount of the rice plasmodesmatal negative regulator OsGSD1 was detected in ossac1 young leaves and developing leaf sheaths than in those of the WT. These results suggest that ER-targeted OsSAC1 may indirectly regulate sugar partitioning in carbon-demanding young leaves and developing leaf sheaths.

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.

In situidentification and quantification of starch-hydrolyzing bacteria attached to barley and corn grain in the rumen of cows fed barley-based diets

Cereal grains rich in starch are widely used to meet the energy demands of high-producing beef and dairy cattle. Bacteria are important players in starch digestion in the rumen, and thus play an important role in the hydrolysis and fermentation of cereal grains. However, our understanding of the composition of the rumen starch-hydrolyzing bacteria (SHB) is limited. In this study, BODIPY FL DQ starch staining combined with fluorescence in situ hybridization (FISH) and quantitative FISH were applied to label, identify and quantify SHB possessing active cell-surface-associated (CSA) α-amylase activity in the rumen of heifers fed barley-based diets. When individual cells of SHB with active CSA α-amylase activity were enumerated, they constituted 19-23% of the total bacterial cells attached to particles of four different cultivars of barley grain and corn. Quantitative FISH revealed that up to 70-80% of these SHB were members of Ruminococcaceae in the phylum Firmicutes but were not Streptococcus bovis, Ruminobacter amylophilus, Succinomonas amylolytica, Bifidobacterium spp. or Butyrivibrio fibrisolvens, all of whose amylolytic activities have been demonstrated previously in vitro. The proportion of barley grain in the diet had a large impact on the percentage abundance of total SHB and Ruminococcaceae SHB in these animals.

Preparation and properties of films cast from mixtures of poly(vinyl alcohol) and submicron particles prepared from amylose-palmitic acid inclusion complexes.

The use of starch in polymer composites for film production has been studied for increasing biodegradability, improving film properties and reducing cost. In this study, submicron particles were prepared from amylose-sodium palmitate complexes both by rapidly cooling jet-cooked starch-palmitic acid mixtures and by acidifying solutions of starch-sodium palmitate complexes. Films were cast containing poly(vinyl alcohol) (PVOH) with up to 50% starch particles. Tensile strength decreased and Young's modulus increased with starch concentration, but percent elongations remained similar to controls regardless of preparation method or starch content. Microscopy showed particulate starch distribution in films made with rapidly cooled starch-palmitic acid particles but smooth, diffuse starch staining with acidified sodium palmitate complexes. The mild effects on tensile properties suggest that submicron starch particles prepared from amylose-palmitic acid complexes provide a useful, commercially viable approach for PVOH film modification.

Heterogeneous soil water flow and macropores described with combined tracers of dye and iodine

Summary The objectives of this study were to examine applicability of the iodine–starch method to visualize heterogenous soil water flow and to investigate relationships between soil water heterogeneity and macropore structure vs. measurement scale as well as hydraulic boundaries. The food-grade dye pigment Brilliant Blue FCF dye and iodine–starch staining patterns were used to visualize soil macropore and soil water flow patterns, respectively. Totally 10 infiltration experiments were performed in the field, among which three were used to examine the iodine–starch method and seven were for various boundary conditions (i.e., the different initial ponding depths on the soil surface) and measurement scales (i.e., the soil surface areas covered initially by the tracer solutions). The cluster analysis method was used to distinguish preferential flow regions, while information measures were applied to quantify heterogeneity information content and complexity of macropores and flow systems. Results showed that the iodine–starch method was applicable to visualize soil water flow. Heterogeneous soil water flow contained more heterogeneity information than soil macropores. With a low infiltration amount, flow patterns were similar to the macropore patterns and controlled mainly by the macropores. As the infiltration amount increased, flow patterns were influenced by both macropores and boundary conditions. As the measurement scales increased, the preferential regions were more developed.

Characterizing soil preferential flow using iodine–starch staining experiments and the active region model

Characterizing Soil Preferential Flow Using Iodine–Starch Staining Experiments and the Active Region Model Hui- Feng Sheng a , Kang Wang b , Renduo Zhang a, and Hui - Hai Liu c a School of Environmental Science and Engineering, Sun Yat-Sen (Zhongshan) University, Guangzhou 510275, China b State Key Lab. of Water Resources and Hydropower Engineering Science, Wuhan University, Wuhan 430072, China c Earth Sciences Division, Lawrence Berkeley National Laboratory, Berkeley, CA, USA

Somatotopic arrangement of sudomotor axon reflexes in human skin

Background: In neuropathies impaired sweating is often one of the first symptoms, therefore the evaluation of the sweating reaction is a valuable method for the early detection of neuropathies. Quantitative sudomotor axon reflex testing (QSART) has been employed for that purpose and measures the amount of sweating induced by iontophoretic stimulation with acetylcholine quantitativelly. For measuring the extent of the sudomotor axon reflex iodine starch staining (Minor testing) has been used. The aim of our study was to compare both tests on three different sites on the leg: foot, lower leg and upper leg.

Somatotopic arrangement of sudomotor axon reflex sweating in humans

Background Impaired sweating may be one of the first symptoms in neuropathies, and therefore the evaluation of sweating might facilitate their early detection. Sudomotor axon reflexes can be quantified by two different methods: quantitative sudomotor axon reflex testing (QSART) measures the amount of local sweating, whereas staining with the iodine starch reaction assesses the extension of the sudomotor axon reflex area. The aim of our study was to compare both tests at three different sites on the leg: foot, lower leg and thigh. Methods QSART and iodine starch staining after iontophoretic stimulation with acetylcholine were performed on 15 male volunteers (mean age: 25; range 24–27 years) on the left resp. the right leg during a single session. Results QSART response, measured as area under the curve (AUC), was maximal at the lower leg (911 AUC), smaller at the dorsum of the foot (585 AUC) and even smaller at the thigh (480 AUC). The difference between lower leg and thigh was significant ( p < 0.02). The sudomotor axon reflex area was also biggest on the lower leg (39 cm 2) followed by the foot dorsum (28 cm 2), and then the thigh (16 cm 2). The differences between lower leg and thigh as well as between lower leg and foot were significant ( p < 0.01, resp. p < 0.04). The size of the sudomotor axon reflex areas and QSART responses were correlated ( p < 0.01). Conclusions QSART and sudomotor axon reflex areas had similar somatotopic arrangements in human skin. The bigger the axon reflex area was the stronger the QSART response was. This indicates that the size of the innervation territories of sudomotor fibres covaries with the amount of local sweat production. The latter is a surrogate for increased sweat gland density or capacity in skin areas of dense sudomotor innervation.

Spreading of sudomotor axon reflexes in human skin

Acetylcholine (ACh) activates both sudomotor fibers and primary afferent nociceptors. This leads to sudomotor and vasodilator axon reflexes, which can be diminished, for example, in neuropathies. In some neuropathies, however, there is increased axon reflex sweating, a response pattern that has never been observed for vasodilator flares. To compare both types of axon reflexes and to elucidate possible differences. In healthy young male subjects, sweat response and flare reaction in response to ACh were quantified. Constant-current iontophoresis (300 mC) of ACh was performed on the lateral lower legs. The sudomotor axon reflex was visualized with iodine starch staining, and the sweat response was quantified with capacitance hygrometry (quantitative sudomotor axon reflex test [QSART]). The vasodilator flare was visualized and quantified by laser Doppler imaging. All measurements were performed during and for 10 minutes after finishing the iontophoresis. The sudomotor axon reflex area increased from 30.6 cm2 at the end of the iontophoresis to 39.2 cm2 (p < 0.001) 10 minutes later, while QSART response had already decreased. Flare size and flare intensity remained nearly constant during the observation period. Despite fast cleavage of acetylcholine by cholinesterases, sudomotor axon reflexes spread in the skin, indicating a possible peripheral amplification of sweating.

Flow path visualization in a lignitic mine soil using iodine–starch staining

A dye tracer visualization experiment was carried out in order to study the importance of preferential flow at forest-reclaimed mine soils. The objective was to evaluate iodine–starch staining for the identification of flow paths in such soils, for the commonly used dye tracer Brilliant Blue showed a poor contrast to the relatively dark lignitic mine soil. The experimental site was located in the Lusatian mining district, northeast Germany, and afforested with Pinus nigra. At the experimental plot area of 250×130 cm, 60 mm of iodide solution was applied at a rate of 7.5 mm/h. Flow path visualization was conducted at 10-cm increments down to 150 cm soil depth. The soil surface was powdered with starch and sprayed with a bleaching agent. Iodine–starch staining indicated the tracer conducting soil regions. Stained soil sections were documented by photography as well as by manual tracing on transparent window films. At each depth increment, 25 core samples were taken at a regular grid of 25×25 cm. For each core sample, the amount of residual iodide was determined. According to the visualized flow paths, the iodide solution infiltrated into the mine soil at distinct points. Beneath a redistribution zone at 10 cm, flow paths occurred as ‘fingers’, which constricted the hydraulically active soil volume to about 15% and persisted down to a depth of 60 cm, at one point to 150 cm. Only a part of the iodine–starch stainings spatially coincided with the vertical iodide concentration. For a closer relationship, the analysis of the complete soil volume seems to be necessary. Iodine–starch staining proved to be suitable for flow path visualization at dark lignitic mine soils under field conditions and seems to be feasible also for other dark soils. At lignitic forest-reclaimed mine soils, fingered flow has to be considered in water and solute budgets.

Dose thresholds and duration of the local anhidrotic effect of botulinum toxin injections: measured by sudometry.

Local injections of botulinum toxin type A (BTX-A) have been used successfully to treat focal hyperhidrosis, but because experimental data were lacking, doses have been chosen arbitrarily or empirically. To analyse dose dependency and duration of BTX-A-derived suppression of sweat gland activity. Employing a standardized scheme (four injections, square 2 x 2 cm), different doses of BTX-A [Dysport(R); 2.5-120 mouse units (MU)] were injected subcutaneously at the lateral aspects of both of the lower legs in 15 healthy volunteers. Sweat tests were performed before, and 3 weeks and 6 months after, BTX-A injections. Sweating was visualized by staining with iodine starch, and quantified by capacitance hygrometry after carbachol iontophoresis, the quantitative sudomotor axon reflex test (QSART). Iodine starch staining indicated a threshold dose of 10 MU (2.5 MU cm-2) leading to visible anhidrotic skin spots after 3 weeks in all subjects. This was maintained for 6 months with doses of 50 MU (12.5 MU cm-2) or higher, but the size of the anhidrotic skin area decreased over time (P < 0.001) indicating partial recovery at the edges. After 3 weeks QSART was significantly reduced (P < 0.02) and completely suppressed by doses of 80 MU (20 MU cm-2) or more, although after 6 months QSART increased again to pre-BTX-A levels (P < 0.001). Both methods indicated that the suppression of sweating is dose dependent (QSART: r = -0.70, P < 0.001; iodine starch staining: r = 0.74, P < 0.001). Our findings suggest that BTX-A effectiveness can be quantified by testing sudomotor function. For the first time threshold doses for the suppression of sweating have been defined.

Sudomotor testing discriminates between subjects with and without antibodies against botulinum toxin A--a preliminary observation.

With an increasing number of patients being treated with botulinum toxin A (BTX A), the incidence of neutralizing anti-BTX antibodies (ABA) is rising. Because BTX A is known to inhibit sweating, sudometry seems to be a promising tool to test the efficacy of BTX A. We injected BTX A subcutaneously in normal control subjects and four patients with spasmodic torticollis, two responders and two nonresponders with proven ABA. Sweating was visualized using iodine starch staining and quantified used capacitance hygrometry. BTX A inhibited sweating completely at the injection site in both control subjects and all responders without evidence for ABA. However, continued sweating was recorded when the nonresponders with proven ABA were tested. We conclude that sudomotor testing is able to discriminate between subjects with and without clinically important ABA.

Spatial extension of sudomotor axon reflex sweating in human skin

Acetylcholine (ACh) applied to human skin is known to elicit a sweat response, which consists of a direct muscarinergic (M3) activation of sweat glands and a nicotinic axon reflex response from sudomotor terminals. To visualize the extent of axon reflex sweating after ACh-iontophoresis, iodine starch staining was used. Iontophoresis was performed under occlusion at the peroneal aspects of the lower leg and the center of the foot dorsum of healthy volunteers ( n=10). Ten minutes after stimulation, the area of dark blue staining was recorded by a video camera. Control experiments were performed with saline, histamine, pilocarpine and nicotine iontophoresis. The stained area was measured and the maximum and minimum distance of its boundary from the edge of the iontophoresis probe was determined (maximum/minimum radius). Sizes of stained areas and maximum radii were significantly greater on the lower leg compared to the foot ( P<0.01). The median sizes of the stained areas on the leg were 14.6 cm 2 and on the foot dorsum 8.0 cm 2. The respective median maximum radii were 3.1 cm on the leg and 2.3 cm on the foot dorsum (median minimum, leg 1.1 cm, foot 0.8 cm). These results match microneurographic findings of innervation territories of sympathetic efferent units. Area sizes of stained skin showed a close correlation between both stimulation sites ( R=0.96, P<0.01), i.e. the sizes of sweat responses on leg and foot show a constant relation (foot/leg=0.57) in spite of their great interindividual variability. This novel technique of establishing sweat responses provides information on the size of sudomotor innervation territories and may be useful for clinical studies in patients with suspected impairment of sympathetic functions.

Isolation and denomination of an important allergen in baking additives: alpha-amylase from Aspergillus oryzae (Asp o II).

The commercially available alpha-amylase from Aspergillus oryzae which is widely used as a baking additive was compared with a highly purified enzyme preparation. We used enzyme allergosorbent test (EAST), EAST inhibition, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focussing, immunoblotting, and N-terminal amino acid sequencing to characterize the causative allergen. Our screening comprised 89 partially selected bakers. Forty-three (48%) of them had work-related respiratory symptoms; 14 (32%) of whom were sensitized to the baking additive. Significant immunological differences could not be found between crude and purified sample with the exception that the latter one produced nearly twice as high antibody values. Iodine starch staining demonstrated that the component which was exclusively or predominantly bound by IgE antibodies of symptomatic bakers represents the active alpha-amylase. According to the International Union of Immunological Societies (IUIS) nomenclature, the term Asp o II is suggested for this important occupational allergen.

A Ripening Index for Banana Fruit Based on Starch Content

A starch staining technique using pictures to rate starch disappearance has been developed to determine banana pulp maturity. The disappearance of starch from the pulp shows linear correlation with peel color (r2 = 0.76) and soluble solids content (r2 = 0.81). Pulp pH shows a poor correlation with starch disappearance (r2 = 0.38). Staining banana pulp starch with an iodine solution is a quick and easy method for estimation of pulp maturity.

Genetic Uniformity in an Introduced Population of Witchweed (Striga asiatica) in the United States

Witchweed, [Striga asiatica(L.) Kuntze5STRLU], an important parasitic weed throughout much of the old world, occurs as a recent (30 yr) introduction in a limited region in North and South Carolina. Reproduction in this population has been shown to occur primarily through self-pollination of flowers. Genetic diversity in two populations from this region was assessed, utilizing starch gel electrophoresis and staining of enzymes. An average of 64 individuals was observed to be monomorphic at an estimated minimum of 32 genetic loci coding 18 enzyme systems. This lack of polymorphism is attributable to the probable founding of the population by one or a few individuals coupled with the predominantly autogamous breeding system.