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Inbred-hybrid breeding of diploid potatoes necessitates breeding lines that are self-compatible. One way of incorporating self-compatibility into incompatible cultivated potato ( Solanum tuberosum ) germplasm is to introduce the S-locus inhibitor gene ( Sli ), which functions as a dominant inhibitor of gametophytic self-incompatibility. To learn more about Sli diversity and function in wild species relatives of cultivated potato, we obtained Sli gene sequences that extended from the 5'UTR to the 3'UTR from 133 individuals from 22 wild species relatives of potato and eight diverse cultivated potato clones. DNA sequence alignment and phylogenetic trees based on genomic and protein sequences show that there are two highly conserved groups of Sli sequences. DNA sequences in one group contain the 533 bp insertion upstream of the start codon identified previously in self-compatible potato. The second group lacks the insertion. Three diploid and four polyploid individuals of wild species collected from geographically disjointed localities contained Sli with the 533 bp insertion. For most of the wild species clones examined, however, Sli did not have the insertion. Phylogenetic analysis indicated that Sli sequences with the insertion, in wild species and in cultivated clones, trace back to a single origin. Some diploid wild potatoes that have Sli with the insertion were self-incompatible and some wild potatoes that lack the insertion were self-compatible. Although there is evidence of positive selection for some codon positions in Sli , there is no evidence of diversifying selection at the gene level. In silico analysis of Sli protein structure did not support the hypothesis that amino acid changes from wild-type (no insertion) to insertion-type account for changes in protein function. Our study demonstrated that genetic factors besides the Sli gene must be important for conditioning a switch in the mating system from self-incompatible to self-compatible in wild potatoes., Competing Interests: The authors declare they have no conflict of interest., (© 2024 The Authors. Plant Direct published by John Wiley & Sons Ltd and American Society of Plant Biologists and the Society for Experimental Biology. This article has been contributed to by U.S. Government employees and their work is in the public domain in the USA.)

Epicotyl length (ECL) of adzuki bean ( Vigna angularis ) affects the efficiency of mechanized weeding and harvest. The present study investigated the genetic factors controlling ECL. An F 2 population derived from a cross between the breeding line 'Tokei1121' (T1121, long epicotyls) and the cultivar 'Erimo167' (common epicotyls) was phenotyped for ECL and genotyped using simple sequence repeats (SSRs) and single-nucleotide polymorphism (SNP) markers. A molecular linkage map was generated and fifty-two segregating markers, including 27 SSRs and 25 SNPs, were located on seven linkage groups (LGs) at a LOD threshold value of 3.0. Four quantitative trait loci (QTLs) for ECL, with LOD scores of 4.0, 3.4, 4.8 and 6.4, were identified on LGs 2, 4, 7 and 10, respectively; together, these four QTLs accounted for 49.3% of the phenotypic variance. The segregation patterns observed in F 5 residual heterozygous lines at qECL10 revealed that a single recessive gene derived from T1121 contributed to the longer ECL phenotype. Using five insertion and deletion markers, this gene was fine mapped to a ~255 kb region near the end of LG10. These findings will facilitate marker-assisted selection for breeding in the adzuki bean and contribute to an understanding of the mechanisms associated with epicotyl elongation., (Copyright © 2021 by JAPANESE SOCIETY OF BREEDING.)

Potato virus Y (PVY) is the most economically important virus infecting potatoes worldwide. Current-season spread of PVY occurs when aphids transmit the virus from infected to noninfected plants during the growing season. The impact of current-season PVY infection on yield and quality of chip processing potatoes is not well documented. In a replicated, greenhouse experiment conducted over 2 years, we measured the effect of current-season infection with four PVY strains (PVY O , PVY N-Wi , PVY NTN , and PVY N:O ) on chip processing varieties Atlantic, Lamoka, and Snowden. PVY infection decreased yield and tuber specific gravity for some combinations of potato variety and virus strain but did not affect the appearance of chips including the prevalence of stem-end chip defects. This work suggests that current-season infection of chipping potatoes imposes a cost on producers and emphasizes the need for continued investment in seed certification and development of PVY-resistant cultivars.

Background: Adverse air and soil temperatures are abiotic stresses that occur frequently and vary widely in duration and magnitude. Heat stress limits productivity of cool-weather crops such as potato (Solanum tuberosum) and may degrade crop quality. Stem-end chip defect is a localized discoloration of potato chips that adversely affects finished chip quality. The causes of stem-end chip defects are poorly understood., Results: Chipping potatoes were grown under controlled environmental conditions to test the hypothesis that stem-end chip defect is caused by transient heat stress during the growing season. Heat stress periods with 35 °C days and 29 °C nights were imposed approximately 3 months after planting and lasted for 3, 7 or 14 days. At harvest and after 1, 2 and 3 months of storage at 13 °C, potato tubers were evaluated for glucose, fructose, sucrose and dry matter contents at the basal and apical ends. Chips were fried and rated for defects at the same sampling times. Differences in responses to heat stress were observed among four varieties of chipping potatoes. Heat stress periods of 7 and 14 days increased reducing sugar content in the tuber basal and apical ends, decreased dry matter content, and increased the severity of stem-end chip defects., Conclusion: Transient heat stress during the growing season decreased post-harvest chipping potato quality. Tuber reducing sugars and stem-end chip defects increased while dry matter content decreased. Planting varieties with tolerance to transient heat stress may be an effective way to mitigate these detrimental effects on chipping potato quality. Published 2018. This article is a U.S. Government work and is in the public domain in the USA., (Published 2018. This article is a U.S. Government work and is in the public domain in the USA.)

Spectral phenotyping is an efficient method for the nondestructive characterization of plant biochemical and physiological status. We examined the ability of a full range (350 to 2,500 nm) of foliar spectral data to (i) detect Potato virus Y (PVY) and physiological effects of the disease in visually asymptomatic leaves, (ii) classify different strains of PVY, and (iii) identify specific potato cultivars. Across cultivars, foliar spectral profiles of PVY-infected leaves were statistically different (F = 96.1, P ≤ 0.001) from noninfected leaves. Partial least-squares discriminate analysis (PLS-DA) accurately classified leaves as PVY infected (validation κ = 0.73) and the shortwave infrared spectral regions displayed the strongest correlations with infection status. Although spectral profiles of different PVY strains were statistically different (F = 6.4, P ≤ 0.001), PLS-DA did not classify different strains well (validation κ = 0.12). Spectroscopic retrievals revealed that PVY infection decreased photosynthetic capacity and increased leaf lignin content. Spectral profiles of potato cultivars also differed (F = 9.2, P ≤ 0.001); whereas average spectral classification was high (validation κ = 0.76), the accuracy of classification varied among cultivars. Our study expands the current knowledge base by (i) identifying disease presence before the onset of visual symptoms, (ii) providing specific biochemical and physiological responses to disease infection, and (iii) discriminating between multiple cultivars within a single plant species.

Background: Genome-wide single nucleotide polymorphism (SNP) markers coupled with allele dosage information has emerged as a powerful tool for studying complex traits in cultivated autotetraploid potato (Solanum tuberosum L., 2n = 4× = 48). To date, this approach has been effectively applied to the identification of quantitative trait loci (QTLs) underlying highly heritable traits such as disease resistance, but largely unexplored for traits with complex patterns of inheritance., Results: In this study, an F 1 tetraploid russet mapping population (162 individuals) was evaluated for multiple quantitative traits over two years and two locations to identify QTLs associated with tuber sugar concentration, processing quality, vine maturity, and other high-value agronomic traits. We report the linkage maps for the 12 potato chromosomes and the QTL location with corresponding genetic models and candidate SNPs explaining the highest phenotypic variation for tuber quality and maturity related traits. Significant QTLs for tuber glucose concentration and tuber fry color were detected on chromosomes 4, 5, 6, 10, and 11. Collectively, these QTLs explained between 24 and 46% of the total phenotypic variation for tuber glucose and fry color, respectively. The QTL on chromosome 10 was associated with apoplastic invertases, with 'Premier Russet' contributing the favorable allele for fry processing quality. On chromosome 5, minor-effect QTLs for tuber glucose concentration and fry color co-localized with various major-effect QTLs, including vine maturity, growth habit, tuber shape, early blight (Altenaria tenuis), and Verticillium wilt (Verticillium spp.)., Conclusions: Linkage analysis and QTL mapping in a russet mapping population (A05141) using SNP dosage information successfully identified favorable alleles and candidate SNPs for resistance to the accumulation of tuber reducing sugars. These novel markers have a high potential for the improvement of tuber processing quality. Moreover, the discovery of different genetic models for traits with overlapping QTLs at the maturity locus clearly suggests an independent genetic control.

As one of the world's most important food crops, the potato ( Solanum tuberosum L.) has spurred innovation in autotetraploid genetics, including in the use of SNP arrays to determine allele dosage at thousands of markers. By combining genotype and pedigree information with phenotype data for economically important traits, the objectives of this study were to (1) partition the genetic variance into additive vs. nonadditive components, and (2) determine the accuracy of genome-wide prediction. Between 2012 and 2017, a training population of 571 clones was evaluated for total yield, specific gravity, and chip fry color. Genomic covariance matrices for additive ( G ), digenic dominant ( D ), and additive × additive epistatic ( G # G ) effects were calculated using 3895 markers, and the numerator relationship matrix ( A ) was calculated from a 13-generation pedigree. Based on model fit and prediction accuracy, mixed model analysis with G was superior to A for yield and fry color but not specific gravity. The amount of additive genetic variance captured by markers was 20% of the total genetic variance for specific gravity, compared to 45% for yield and fry color. Within the training population, including nonadditive effects improved accuracy and/or bias for all three traits when predicting total genotypic value. When six F 1 populations were used for validation, prediction accuracy ranged from 0.06 to 0.63 and was consistently lower (0.13 on average) without allele dosage information. We conclude that genome-wide prediction is feasible in potato and that it will improve selection for breeding value given the substantial amount of nonadditive genetic variance in elite germplasm., (Copyright © 2018 by the Genetics Society of America.)

Background: Potato chip processors require potato tubers that meet quality specifications for fried chip color, and color depends largely upon tuber sugar contents. At later times in storage, potatoes accumulate sucrose, glucose, and fructose. This developmental process, senescent sweetening, manifests as a blush of color near the center of the fried chip, becomes more severe with time, and limits the storage period. Vacuolar invertase (VInv) converts sucrose to glucose and fructose and is hypothesized to play a role in senescent sweetening. To test this hypothesis, senescent sweetening was quantified in multiple lines of potato with reduced VInv expression., Results: Chip darkening from senescent sweetening was delayed by about 4 weeks for tubers with reduced VInv expression. A strong positive correlation between frequency of dark chips and tuber hexose content was observed. Tubers with reduced VInv expression had lower hexose to sucrose ratios than controls., Conclusion: VInv activity contributes to reducing sugar accumulation during senescent sweetening. Sucrose breakdown during frying may contribute to chip darkening. Suppressing VInv expression increases the storage period of the chipping potato crop, which is an important consideration, as potatoes with reduced VInv expression are entering commercial production in the USA. © 2017 Society of Chemical Industry., (© 2017 Society of Chemical Industry.)

Key Message: New software to make tetraploid genotype calls from SNP array data was developed, which uses hierarchical clustering and multiple F1 populations to calibrate the relationship between signal intensity and allele dosage. SNP arrays are transforming breeding and genetics research for autotetraploids. To fully utilize these arrays, the relationship between signal intensity and allele dosage must be calibrated for each marker. We developed an improved computational method to automate this process, which is provided as the R package ClusterCall. In the training phase of the algorithm, hierarchical clustering within an F1 population is used to group samples with similar intensity values, and allele dosages are assigned to clusters based on expected segregation ratios. In the prediction phase, multiple F1 populations and the prediction set are clustered together, and the genotype for each cluster is the mode of the training set samples. A concordance metric, defined as the proportion of training set samples equal to the mode, can be used to eliminate unreliable markers and compare different algorithms. Across three potato families genotyped with an 8K SNP array, ClusterCall scored 5729 markers with at least 0.95 concordance (94.6% of its total), compared to 5325 with the software fitTetra (82.5% of its total). The three families were used to predict genotypes for 5218 SNPs in the SolCAP diversity panel, compared with 3521 SNPs in a previous study in which genotypes were called manually. One of the additional markers produced a significant association for vine maturity near a well-known causal locus on chromosome 5. In conclusion, when multiple F1 populations are available, ClusterCall is an efficient method for accurate, autotetraploid genotype calling that enables the use of SNP data for research and plant breeding.

Acrylamide is produced in a wide variety of carbohydrate-rich foods during high-temperature cooking. Dietary acrylamide is a suspected human carcinogen, and health concerns related to dietary acrylamide have been raised worldwide. French fries and potato chips contribute a significant proportion to the average daily intake of acrylamide, especially in developed countries. One way to mitigate health concerns related to acrylamide is to develop potato cultivars that have reduced contents of the acrylamide precursors asparagine, glucose and fructose in tubers. We generated a large number of silencing lines of potato cultivar Russet Burbank by targeting the vacuolar invertase gene VInv and the asparagine synthetase genes StAS1 and StAS2 with a single RNA interference construct. The transcription levels of these three genes were correlated with reducing sugar (glucose and fructose) and asparagine content in tubers. Fried potato products from the best VInv/StAS1/StAS2-triple silencing lines contained only one-fifteenth of the acrylamide content of the controls. Interestingly, the extent of acrylamide reduction of the best triple silencing lines was similar to that of the best VInv-single silencing lines developed previously from the same potato cultivar Russet Burbank. These results show that an acrylamide mitigation strategy focused on developing potato cultivars with low reducing sugars is likely to be an effective and sufficient approach for minimizing the acrylamide-forming potential of French fry processing potatoes., (© 2015 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Storing potato tubers at low temperatures minimizes sprouting and disease but can cause an accumulation of reducing sugars in a process called cold-induced sweetening. Tubers with increased amounts of reducing sugars produce dark-colored, bitter-tasting fried products with elevated amounts of acrylamide, a possible carcinogen. Vacuolar invertase (VInv), which converts sucrose produced by starch breakdown to glucose and fructose, is the key determinant of reducing sugar accumulation during cold-induced sweetening. In this study, wild-type tubers and tubers in which VInv expression was reduced by RNA interference were used to investigate time- and temperature-dependent changes in sugar contents, chip color, and expression of VInv and other genes involved in starch metabolism in tubers during long-term cold storage., Results: VInv activities and tuber reducing sugar contents were much lower, and tuber sucrose contents were much higher, in transgenic than in wild-type tubers stored at 3-9°C for up to eight months. Large differences in VInv mRNA accumulation were not observed at later times in storage, especially at temperatures below 9°C, so differences in invertase activity were likely established early in the storage period and maintained by stability of the invertase protein. Sugar contents, chip color, and expression of several of the studied genes, including AGPase and GBSS, were affected by storage temperature in both wild-type and transgenic tubers. Though transcript accumulation for other sugar-metabolism genes was affected by storage temperature and duration, it was essentially unaffected by invertase silencing and altered sugar contents. Differences in stem- and bud-end sugar contents in wild-type and transgenic tubers suggested different compartmentalization of sucrose at the two ends of stored tubers., Conclusions: VInv silencing significantly reduced cold-induced sweetening in stored potato tubers, likely by means of differential VInv expression early in storage. Transgenic tubers retained sensitivity to storage temperature, and accumulated greater amounts of sucrose, glucose and fructose at 3°C than at 7-9°C. At each storage temperature, suppression of VInv expression and large differences in tuber sugar contents had no effect on expression of AGPase and GBSS, genes involved in starch metabolism, suggesting that transcription of these genes is not regulated by tuber sugar content.

Sugar-end defect is a tuber quality disorder and persistent problem for the French fry processing industry that causes unacceptable darkening of one end of French fries. This defect appears when environmental stress during tuber growth increases post-harvest vacuolar acid invertase activity at one end of the tuber. Reducing sugars produced by invertase form dark-colored Maillard reaction products during frying. Acrylamide is another Maillard reaction product formed from reducing sugars and acrylamide consumption has raised health concerns worldwide. Vacuolar invertase gene (VInv) expression was suppressed in cultivars Russet Burbank and Ranger Russet using RNA interference to determine if this approach could control sugar-end defect formation. Acid invertase activity and reducing sugar content decreased at both ends of tubers. Sugar-end defects and acrylamide in fried potato strips were strongly reduced in multiple transgenic potato lines. Thus vacuolar invertase silencing can minimize a long-standing French fry quality problem while providing consumers with attractive products that reduce health concerns related to dietary acrylamide.

The ubiquitous signaling molecule nitric oxide (NO) plays an important role in seed biology. Experiments with this biologically important gas require special provisions because NO in aerobic environments is readily converted into other oxides of nitrogen. In this chapter, we describe methods for the application of NO as a gas, and through the use of NO-donor compounds. We included information on the removal or reduction of NO with NO scavengers. Methods for detecting NO using NO-reactive fluorescent probes, and an apparatus incorporating an oxidizer column are also described.

Potato (Solanum tuberosum) is the third most important food crop in the world. Potato tubers must be stored at cold temperatures to prevent sprouting, minimize disease losses, and supply consumers and the processing industry with high-quality tubers throughout the year. Unfortunately, cold storage triggers an accumulation of reducing sugars in tubers. High-temperature processing of these tubers results in dark-colored, bitter-tasting products. Such products also have elevated amounts of acrylamide, a neurotoxin and potential carcinogen. We demonstrate that silencing the potato vacuolar acid invertase gene VInv prevents reducing sugar accumulation in cold-stored tubers. Potato chips processed from VInv silencing lines showed a 15-fold acrylamide reduction and were light in color even when tubers were stored at 4°C. Comparable, low levels of VInv gene expression were observed in cold-stored tubers from wild potato germplasm stocks that are resistant to cold-induced sweetening. Thus, both processing quality and acrylamide problems in potato can be controlled effectively by suppression of the VInv gene through biotechnology or targeted breeding.

Cold-induced sweetening and browning in the Maillard reaction have driven extensive research in the areas of plant physiology, biochemistry, and food science in Solanum tuberosum because of its importance to the potato-processing industry. Prior research has not characterized wild Solanum relatives of potato for tuber composition and has not determined if relationships between tuber composition and chip color after cold storage in wild species are comparable to those found for cultivated potato. Extensive inter- and intraspecific variation for chip color and tuber composition were found in the wild Solanum species examined. Tuber sugar profiles suggested that invertase activity at low temperatures differed between and within species. Tuber fructose, glucose, and sucrose concentrations partially explained chip color variation in most accessions, but asparagine concentration and percent dry matter did not. Most wild species had reducing sugar concentrations and chip color scores after 2 degrees C storage that were less than those in S. tuberosum cultivar Snowden. Sugar profiles and relationships between specific sugars and chip color in Solanum pinnatisectum were unique among the species examined.

The white potato (Solanum tuberosum L.) is a valuable source of potassium in the human diet. While most consumers benefit from high levels of potassium in potato tubers, individuals with compromised kidney function must minimize their potassium intake. This study was undertaken to determine the effects of leaching and boiling on levels of potassium and other minerals in potato tubers. Leaching alone did not significantly reduce levels of potassium or other minerals in tubers. Boiling tuber cubes and shredded tubers decreased potassium levels by 50% and 75%, respectively. Reductions in mineral amounts following boiling were observed for phosphorus, magnesium, sulfur, zinc, manganese, and iron. There was no difference between the leaching and boiling treatment and the boiling treatment. In addition, mineral levels in tubers of 6 North American potato cultivars are reported. Significant differences in mineral levels were detected among cultivars, but they were too small to be nutritionally important. Individuals wishing to maximize the mineral nutrition benefits of consuming potatoes should boil them whole or bake, roast, or microwave them. Those who must reduce potassium uptake should boil small pieces before consuming them.

Seed dormancy is a common phase of the plant life cycle, and several parts of the seed can contribute to dormancy. Whole seeds, seeds lacking the testa, embryos, and isolated aleurone layers of Arabidopsis (Arabidopsis thaliana) were used in experiments designed to identify components of the Arabidopsis seed that contribute to seed dormancy and to learn more about how dormancy and germination are regulated in this species. The aleurone layer was found to be the primary determinant of seed dormancy. Embryos from dormant seeds, however, had a lesser growth potential than those from nondormant seeds. Arabidopsis aleurone cells were examined by light and electron microscopy, and cell ultrastructure was similar to that of cereal aleurone cells. Arabidopsis aleurone cells responded to nitric oxide (NO), gibberellin (GA), and abscisic acid, with NO being upstream of GA in a signaling pathway that leads to vacuolation of protein storage vacuoles and abscisic acid inhibiting vacuolation. Molecular changes that occurred in embryos and aleurone layers prior to germination were measured, and these data show that both the aleurone layer and the embryo expressed the NO-associated gene AtNOS1, but only the embryo expressed genes for the GA biosynthetic enzyme GA3 oxidase.

The nitric oxide (NO) donor sodium nitroprusside (SNP) significantly promoted germination of switchgrass (Panicum virgatum L. cv Kanlow) in the light and in the dark at 25 degrees C, across a broad range of concentrations. SNP also promoted seed germination in two other warm-season grasses. A chemical scavenger of NO inhibited germination and blocked SNP stimulation of seed germination. The phenolic (+)-catechin acted synergistically with SNP and nitrite in promoting seed germination. Acidified nitrite, an alternate NO donor also significantly stimulated seed germination. Interestingly, sodium cyanide, potassium ferricyanide and potassium ferrocyanide at 200 microM strongly enhanced seed germination as well, whereas potassium chloride was without effect. Ferrocyanide and cyanide stimulation of seed germination was blocked by an NO scavenger. Incubation of seeds with a fluorescent NO-specific probe provided evidence for NO production in germinating switchgrass seeds. Abscisic acid (ABA) at 10 microM depressed germination, inhibited root elongation and essentially abolished coleoptile emergence. SNP partially overcame ABA effects on radicle emergence but did not overcome the effects of ABA on coleoptile elongation. Light microscopy indicated extension of the radicle and coleoptiles in seeds maintained on water or on SNP after 2 days. In contrast, there was minimal growth of the radicle and coleoptile in ABA-treated seeds even after 3-4 days. These data indicate that seed germination of warm-season grasses is significantly influenced by NO signaling pathways and document that NO could be an endogenous trigger for release from dormancy in these species.

The seeds of many plant species are dormant at maturity and dormancy loss is a prerequisite for germination. Numerous environmental and chemical treatments are known to lessen or remove seed dormancy, but the biochemical changes that occur during this change of state are poorly understood. Several lines of research have implicated nitric oxide (NO) as a participant in this process. Here, we show that dormant seeds of Arabidopsis thaliana (L.) Heynh. will germinate following treatment with the NO donor sodium nitroprusside (SNP), cyanide (CN), nitrite or nitrate. In all cases, the NO scavenger c-PTIO effectively promotes the maintenance of seed dormancy. c-PTIO does not, however, inhibit germination of fully after-ripened seeds, and c-PTIO does not interact directly with nitrite, nitrate or CN. We also show that volatile CN effectively breaks dormancy of Arabidopsis seeds, and that CN is the volatile compound in SNP that promotes dormancy loss. Our data support the hypothesis that NO is a signaling molecule that plays an important role in the loss of seed dormancy.

The cereal aleurone layer is a model system for studying the regulation of transcription by gibberellin (GA) and abscisic acid (ABA). GA stimulates and ABA prevents the transcription of genes for alpha-amylases and other secreted hydrolytic enzymes, but how GA and ABA affect the transcription of other genes is largely unknown. We characterized gene expression in rice (Oryza sativa) aleurone using a half-genome rice microarray. Of the 23,000 probe sets on the chip, approximately 11,000 hybridized with RNA from rice aleurone treated with ABA, GA, or no hormone. As expected, GA regulated the expression of many genes, and 3 times as many genes were up-regulated by GA at 8 h than were down-regulated. Changes in gene expression resulting from ABA treatment were not consistent with the hypothesis that the role of ABA in this tissue is primarily to repress gene expression, and 10 times more genes were up-regulated by ABA at 8 h than were down-regulated by ABA. We also measured transcript abundance in aleurone of dwarf1 (d1) mutant rice. The d1 protein is the sole alpha-subunit of heterotrimeric G-proteins in rice. Genes up-regulated by GA or ABA had higher expression in wild type than in d1 aleurone, and genes down-regulated by GA had lower expression in wild type relative to d1 aleurone. The d1 mutation did not result in a decrease in sensitivity to GA at the level of transcription. Rather, changes in transcript abundance were smaller in the d1 mutant than in wild type.

Dormancy is a property of many mature seeds, and experimentation over the past century has identified numerous chemical treatments that will reduce seed dormancy. Nitrogen-containing compounds including nitrate, nitrite, and cyanide break seed dormancy in a range of species. Experiments are described here that were carried out to further our understanding of the mechanism whereby these and other compounds, such as the nitric oxide (NO) donor sodium nitroprusside (SNP), bring about a reduction in seed dormancy of Arabidopsis thaliana. A simple method was devised for applying the products of SNP photolysis through the gas phase. Using this approach it was shown that SNP, as well as potassium ferricyanide (Fe(III)CN) and potassium ferrocyanide (Fe(II)CN), reduced dormancy of Arabidopsis seeds by generating cyanide (CN). The effects of potassium cyanide (KCN) on dormant seeds were tested and it was confirmed that cyanide vapours were sufficient to break Arabidopsis seed dormancy. Nitrate and nitrite also reduced Arabidopsis seed dormancy and resulted in substantial rates of germination. The effects of CN, nitrite, and nitrate on dormancy were prevented by the NO scavenger c-PTIO. It was confirmed that NO plays a role in reducing seed dormancy by using purified NO gas, and a model to explain how nitrogen-containing compounds may break dormancy in Arabidopsis is presented.

Many developmental and environmental signals are transduced through changes in intracellular calcium concentrations, yet only a few calcium-binding proteins have been identified in plants. Calcineurin B-like (CBL) proteins are calcium-binding proteins that are thought to function as plant signal transduction elements. RNA profiling using a rice (Oryza sativa cv Nipponbare) oligonucleotide microarray was used to monitor gene expression in de-embryonated rice grains. This analysis showed that a putative rice CBL gene responded to gibberellic acid, but not abscisic acid, treatment. The CBL gene family in rice contains at least 10 genes and these have extensive similarity to the CBLs of Arabidopsis (Arabidopsis thaliana). In yeast (Saccharomyces cerevisiae) two-hybrid assays, rice CBLs interact with the kinase partners of Arabidopsis CBLs. Only one rice CBL gene, OsCBL2, is up-regulated by GA in the aleurone layer. A homolog with 91% sequence identity to OsCBL2 was cloned from barley (Hordeum vulgare cv Himalaya), and designated HvCBL2. We examined the localization and function of OsCBL2 and HvCBL2 in rice and barley aleurone because changes in cytosolic calcium have been implicated in the response of the aleurone cell to GA. Green fluorescent protein translational fusions of OsCBL2 and OsCBL3 were localized to the tonoplast of aleurone cell protein storage vacuoles and OsCBL4-green fluorescent protein was localized to the plasma membrane. Data from experiments using antisense expression of OsCBL2 and HvCBL2 are consistent with a role for OsCBL2 in promoting vacuolation of barley aleurone cells following treatment with GA.

Seeds of Arabidopsis thaliana (L.) Heynh. and grains of barley ( Hordeum vulgare L.) were used to characterize the affects of nitric oxide (NO) on seed dormancy. Seeds of the C24 and Col-1 ecotypes of Arabidopsis are almost completely dormant when freshly harvested, but dormancy was broken by stratification for 3 days at 4 degrees C or by imbibition of seeds with the NO donor sodium nitroprusside (SNP). This effect of SNP on dormancy of Arabidopsis seeds was concentration dependent. SNP concentrations as low as 25 microM reduced dormancy and stimulated germination, but SNP at 250 microM or more impaired seedling development, including root growth, and inhibited germination. Dormancy was also reduced when Arabidopsis seeds were exposed to gases that are generated by solutions of SNP. Nitrate and nitrite, two other oxides of nitrogen, reduced the dormancy of Arabidopsis seeds, but much higher concentrations of these were required compared to SNP. Furthermore, the kinetics of germination were slower for seeds imbibed with either nitrate or nitrite than for seeds imbibed with SNP. Although seeds imbibed with SNP had reduced dormancy, seeds imbibed with SNP and abscisic acid (ABA) remained strongly dormant. This may indicate that the effects of ABA action on germination are downstream of NO action. The NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3 oxide (cPTIO) strengthened dormancy of unstratified and briefly stratified Arabidopsis seeds. Dormancy of three cultivars of barley was also reduced by SNP. Furthermore, dormancy in barley grain was strengthened by imbibition of grain with cPTIO. The data presented here support the conclusion that NO is a potent dormancy breaking agent for seeds and grains. Experiments with the NO scavenger suggest that NO is an endogenous regulator of seed dormancy.

Nitric oxide (NO) is an important signaling molecule in animals and plants. In mammals, NO is produced from Arg by the enzyme NO synthase. In plants, NO synthesis from Arg using an NO synthase-type enzyme and from nitrite using nitrate reductase has been demonstrated previously. The data presented in this report strongly support the hypothesis that plant tissues also synthesize NO via the nonenzymatic reduction of apoplastic nitrite. As measured by mass spectrometry or an NO-reactive fluorescent probe, Hordeum vulgare (barley) aleurone layers produce NO rapidly when nitrite is added to the medium in which they are incubated. NO production requires an acid apoplast and is accompanied by a loss of nitrite from the medium. Phenolic compounds in the medium can increase the rate of NO production. The possible significance of apoplastic NO production for germinating grain and for plant roots is discussed.

The putative H+/Cl- symporter cycloprodigiosin-HCl (cPrG-HCl) was used to investigate the role of vacuole acidification in cereal aleurone cell function. The protein storage vacuole (PSV) becomes acidified rapidly when aleurone cells are treated with gibberellic acid (GA) but not abscisic acid (ABA). We show that cPrG prevents PSV acidification in aleurone layers and prevents synthesis of secretory proteins such as alpha-amylase. Our data support the hypothesis that decreased hydrolase synthesis is a consequence of decreased hydrolysis of storage proteins in PSV. Support for this hypothesis comes from experiments showing that breakdown of barley 7S globulins and phytate is inhibited by cPrG in GA-treated aleurone layers. Decreased mobilization of PSV reserves is accompanied by reductions in the free amino acid pool size and in the amount of ions released from the aleurone layer. Vacuolation of the aleurone cell is a diagnostic feature of the response to GA, and vacuolation is also inhibited by cPrG. Evidence that cPrG acts as a potential H+/Cl- symporter in aleurone is presented. We show that cPrG does not inhibit the synthesis and secretion of alpha-amylase when Cl- ions are omitted from the incubation medium. Although cPrG blocks many GA-induced responses of aleurone layers, it does not affect early steps in GA signaling. The SLN1 protein, a negative regulator of GA signaling, is turned over in GA-treated cells in the presence and absence of cPrG. Similarly, synthesis of the transcriptional activator GAMYB is unaffected by the presence of cPrG in GA-treated cells.

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

The cereal aleurone is widely used as a model system to study hormonal signalling. Abscisic acid (ABA) and gibberellins (GAs) elicit distinct responses in aleurone cells, ranging from those occurring within minutes of hormone addition to those that require several hours or days to complete. Programmed cell death is an example of a response in aleurone layers that is hormonally regulated. GAs promote cell death and cells in intact aleurone layers begin to die 24 h after GA treatment, whereas cell death of aleurone protoplasts begins 4 d after GA treatment. ABA prevents aleurone cell death and addition of ABA to cells pretreated with GA can delay cell death. Aleurone cells do not follow the apoptotic route of programmed cell death. Cells treated with GA, but not ABA, develop large, acidic vacuoles containing a spectrum of hydrolases typical of lytic compartments. Enzymes that metabolize reactive oxygen species are also present in aleurone cells, but ascorbate peroxidase, catalase and superoxide dismutase become less abundant after treatment with GA; activity of these enzymes increases or remains unchanged in ABA-treated cells. We propose a model whereby reactive oxygen species accumulate in GA-treated cells and lead to peroxidation of membrane lipids and plasma membrane rupture. ABBREVIATIONS: RO, reactive oxygen species; HR, hypersensitive response; PSV, protein storage vacuole; PCD, programmed cell death; CAT, catalase; SOD, superoxide dismutase; APX, ascorbate peroxidase.

Gibberellins (GAs) initiate a series of events that culminate in programmed cell death, whereas abscisic acid (ABA) prevents this process. Reactive oxygen species (ROS) are key elements in aleurone programmed cell death. Incubation of barley (Hordeum vulgare) aleurone layers in H2O2 causes rapid death of all cells in GA- but not ABA-treated layers. Sensitivity to H2O2 in GA-treated aleurone cells results from a decreased ability to metabolize ROS. The amounts and activities of ROS scavenging enzymes, including catalase (CAT), ascorbate peroxidase, and superoxide dismutase are strongly down-regulated in aleurone layers treated with GA. CAT activity, protein, and Cat2 mRNA decline rapidly following exposure of aleurone layers to GA. In ABA-treated layers, on the other hand, the amount and activity of CAT and Cat2 mRNA increases. Incubation in ABA maintains high amounts of ascorbate peroxidase and superoxide dismutase, whereas GA brings about a rapid reduction in the amounts of these enzymes. These data imply that GA-treated cells loose their ability to scavenge ROS and that this loss ultimately results in oxidative damage and cell death. ABA-treated cells, on the other hand, maintain their ability to scavenge ROS and remain viable.

The barley aleurone layer is a terminally differentiated secretory tissue whose activity is hormonally controlled. The plant hormone gibberellic acid (GA) stimulates the secretion of hydrolytic enzymes and triggers the onset of programmed cell death (PCD). Abscisic acid (ABA) antagonizes the effects of GA and inhibits enzyme secretion and PCD. Reactive oxygen species (ROS) are key players in many types of PCD, and data presented here implicate ROS in hormonally regulated death of barley aleurone cells. Incubation of aleurone layers or protoplasts in H(2)O(2)-containing media results in death of GA-treated but not ABA-treated aleurone cells. Cells that are programmed to die are therefore less able to withstand ROS than cells that are programmed to remain alive. Illumination of barley aleurone protoplasts with blue or UV-A light results in a rapid increase in intracellular H(2)O(2) production. GA-treated protoplasts die rapidly in response to this increase in intracellular H(2)O(2) production, but ABA-treated protoplasts do not die. The rate of light-induced death could be slowed by antioxidants, and incubating protoplasts in the dark with the antioxidant butylated hydroxy toluene reduces the rate of hormonally induced death. Taken together, these data demonstrate that GA-treated aleurone protoplasts are less able than ABA-treated protoplasts to tolerate internally generated or exogenously applied H(2)O(2), and strongly suggest that ROS are components of the hormonally regulated cell death pathway in barley aleurone cells.

Plant vacuoles are complex and dynamic organelles. Important advances have been made in our understanding of the transporters present in the tonoplast and of the molecular interactions that allow targeting to vacuoles. Despite these advances, markers that permit vacuoles to be defined unambiguously have not yet been identified.

Barley aleurone cells undergo programmed cell death (PCD) when exposed to gibberellic acid (GA), but incubation in abscisic acid (ABA) prevents PCD. We tested the hypothesis that PCD in aleurone cells occurs by apoptosis, and show that the hallmarks of apoptosis, namely DNA cleavage into 180 bp fragments, plasma membrane blebbing, and the formation of apoptotic bodies do not occur when aleurone cells die. We show that endogenous barley aleurone nucleases and nucleases present in enzymes used for protoplast preparation degrade aleurone DNA and that DNA degradation by these nucleases is rapid and can result in the formation of 180 bp DNA ladders. Methods are described that prevent DNA degradation during isolation from aleurone layers or protoplasts. Barley aleurone cells contain three nucleases whose activities are regulated by GA and ABA. GA induction and ABA repression of nuclease activities correlate with PCD in aleurone cells. Cells incubated in ABA remain alive and do not degrade their DNA, but living aleurone cells treated with GA accumulate nucleases and hydrolyze their nuclear DNA. We propose that barley nucleases play a role in DNA cleavage during aleurone PCD.

Cell death was studied in barley (cv Himalaya) aleurone cells treated with abscisic acid and gibberellin. Aleurone protoplasts incubated in abscisic acid remained viable in culture for at least 3 weeks, but exposure to gibberellin initiated a series of events that resulted in death. Between 4 and 8 days after incubation in gibberellin, >70% of all protoplasts died. Death, which occurred after cells became highly vacuolated, was manifest by an abrupt loss of plasma membrane integrity followed by rapid shrinkage of the cell corpse. Hydrolysis of DNA began before death and occurred as protoplasts ceased production of alpha-amylase. DNA degradation did not result in the accumulation of discrete low molecular weight fragments. DNA degradation and cell death were prevented by LY83583, an inhibitor of gibberellin signaling in barley aleurone. We conclude that cell death in aleurone cells is hormonally regulated and is the final step of a developmental program that promotes successful seedling establishment.

Recent findings provide insights into the gibberellin signaling system in plants. Genes for gibberellin biosynthetic enzymes have been cloned, and an emerging theme is that gibberellin biosynthesis is negatively regulated by gibberellin responses. Mutants defective in gibberellin signaling have been analyzed, and an important finding is that gibberellin represses growth inhibition. The list of intracellular gibberellin signal-transduction elements has been expanded to include G-proteins and protein kinases.

Light microscopy was used to study the structure and function of vacuoles in living protoplasts of barley (Hordeum vulgare cv Himalaya) aleurone. Light microscopy showed that aleurone protoplasts contain two distinct types of vacuole: the protein storage vacuole and a lysosome-like organelle, which we have called the secondary vacuole. Fluorescence microscopy using pH-sensitive fluorescent probes and a fluorogenic substrate for cysteine proteases showed that both protein storage vacuoles and secondary vacuoles are acidic, lytic organelles. Ratio imaging showed that the pH of secondary vacuoles was lower in aleurone protoplasts incubated in gibberellic acid than in those incubated in abscisic acid. Uptake of fluorescent probes into intact, isolated protein storage vacuoles and secondary vacuoles required ATP and occurred via at least two types of vanadate-sensitive, ATP-dependent tonoplast transporters. One transporter catalyzed the accumulation of glutathione-conjugated probes, and another transported probes not conjugated to glutathione.

Within the cereal aleurone reserve proteins are stored in specialized organelles, the protein storage vacuoles (PSV). We developed an aqueous method for the isolation of intact PSV. Barley (Hordeum vulgare L. cv Himalaya) aleurone protoplasts were gently lysed by passing them through a syringe needle. PSV were separated from cytoplasmic components by microfiltration and low-speed centrifugation. Isolated PSV appeared by light microscopy to be identical with those within barley aleurone protoplasts. Luminal contents were retained throughout the isolation procedure. We used isolated PSV to identify and characterize PSV-associated proteolytic activities. Isolated PSV contained cysteine proteases and aspartic proteases (APs). Gibberellic acid treatment of protoplasts increased cysteine protease activity. Protein blots probed with anti-H. vulgare aspartic proteinase (HvAP) indicated that one PSV-AP was HvAP. Immunocytochemical localization by electron microscopy confirmed the presence of HvAP within the lumen of PSV. We conclude that isolated barley aleurone PSV will be useful in further characterizing this organelle.

Many plant ion channels have been identified, but little is known about how these transporters are regulated. We have investigated the regulation of a slow vacuolar (SV) ion channel in the tonoplast of barley aleurone storage protein vacuoles (SPV) using the patch-clamp technique. SPV were isolated from barley aleurone protoplasts incubated with CaCl2 in the presence or absence of gibberellic acid (GA) or abscisic acid (ABA). A slowly activating, voltage-dependent ion channel was identified in the SPV membrane. Mean channel conductance was 26 pS when 100 mM KCl was on both sides of the membrane, and reversal potential measurements indicated that most of the current was carried by K+. Treatment of protoplasts with GA3 increased whole-vacuole current density compared to SPV isolated from ABA- or CaCl2-treated cells. The opening of the SV channel was sensitive to cytosolic free Ca2+ concentration ([Ca2+]i) between 600 nM and 100 [mu]M, with higher [Ca2+]i resulting in a greater probability of channel opening. SV channel activity was reduced greater than 90% by the calmodulin (CaM) inhibitors W7 and trifluoperazine, suggesting that Ca2+ activates endogenous CaM tightly associated with the membrane. Exogenous CaM partially reversed the inhibitory effects of W7 on SV channel opening. CaM also sensitized the SV channel to Ca2+. In the presence of ~3.5 [mu]M CaM, specific current increased by approximately threefold at 2.5 [mu]M Ca2+ and by more than 13-fold at 10 [mu]M Ca2+. Since [Ca2+]i and the level of CaM increase in barley aleurone cells following exposure to GA, we suggest that Ca2+ and CaM act as signal transduction elements mediating hormone-induced changes in ion channel activity.

Young bell pepper (Capsicum annuum L.) plants grown in nutrient solution were gradually acclimated to 50, 100, or 150 moles per cubic meter NaCl, and photosynthetic rates of individual attached leaves were measured on several occasions during the salinization period at external CO(2) concentrations ranging from approximately 70 to 1900 micromoles per mole air. Net CO(2) assimilation (A) was plotted against computed leaf internal CO(2) concentration (C(i)), and the initial slope of this A-C(i) curve was used as a measure of photosynthetic ability. During the 10 to 14 days after salinization began, leaves from plants exposed to 50 moles per cubic meter NaCl showed little change in photosynthetic ability, whereas those treated to 100 or 150 moles per cubic meter NaCl had up to 85% inhibition, with increase in CO(2) compensation point. Leaves appeared healthy, and leaf chlorophyll content showed only a 14% reduction at the highest salinity levels. Partial stomatal closure occurred with salinization, but reductions in photosynthesis were primarily nonstomatal in origin. Photosynthetic ability was inversely related to the concentration of either Na(+) or Cl(-) in the leaf laminas sampled at the end of the experimental period. However, the concentration of Cl(-) expressed on a tissue water basis was greater, exceeding 300 moles per cubic meter, and Cl(-) was more closely associated (R(2) = 0.926) with the inhibition of photosynthetic ability. Leaf turgor was not reduced by salinization and leaf osmotic potential decreased to a slightly greater extent than the osmotic potential decreases of the nutrient solutions. Concentration of accumulated Na(+) and Cl(-) (on a tissue water basis) accounted quantitatively for maintenance of leaf osmotic balance, assuming that these ions were sequestered in the vacuoles.