Temperature ( T ) and water potential ( y ) are two primary environmental regulators of seed germination. Seeds exhibit a base or minimum T for germination ( T b ), an optimum T at which germination is most rapid ( T o ), and a maximum or ceiling T at which germination is prevented ( T c ). Germination at suboptimal T can be characterized on the basis of thermal time, or the T in excess of T b multiplied by the time to a given germination percentage ( t g ). Similarly, germination at reduced y can be characterized on a hydrotime basis, or t g multiplied by the y in excess of a base or threshold y that just prevents germination ( y b ). Within a seed population, the variation in thermal times to germination among different seed fractions ( g ) is based on a normal distribution of y b values among seeds ( y b ( g )). Germination responses across a range of suboptimal T and y can be described by a general hydrothermal time model that combines the T and y components, but this model does not account for the decrease in germination rates and percentages when T exceeds T o . We report here that supra-optimal temperatures shift the ψ ψ ψ ψ b ( g ) distribution of a potato ( Solanum tuberosum L.) seed population to more positive values, explaining why both germination rates and percentages are reduced as T increases above T o . A modified hydrothermal time model incorporating changes in ψ ψ ψ ψ b ( g ) at T > T o describes germination timing and percentage across all T and ψ ψ ψ ψ at which germination can occur and provides physiologically relevant indices of seed behaviour.

.)Raffinose family oligosaccharides (RFOs) have been implicated in mitigating the effects of environmental stresses on plants. In seeds, proposed roles for RFOs include protecting cellular integrity during desiccation and/or imbibition, extending longevity in the dehydrated state, and providing substrates for energy generation during germination. A gene encoding galactinol synthase (GOLS), the first committed enzyme in the biosynthesis of RFOs, was cloned from tomato (Lycopersicon esculentum Mill. cv Moneymaker) seeds, and its expression was characterized in tomato seeds and seedlings. GOLS (LeGOLS-1) mRNA accumulated in developing tomato seeds concomitant with maximum dry weight deposition and the acquisition of desiccation tolerance. LeGOLS-1 mRNA was present in mature, desiccated seeds but declined within 8 h of imbibition in wild-type seeds. However, LeGOLS-1 mRNA accumulated again in imbibed seeds prevented from completing germination by dormancy or water deficit. Gibberellin-deficient (gib-1) seeds maintained LeGOLS-1 mRNA amounts after imbibition unless supplied with gibberellin, whereas abscisic acid (ABA) did not prevent the loss of LeGOLS-1 mRNA from wild-type seeds. The presence of LeGOLS-1 mRNA in ABA-deficient (sitiens) tomato seeds indicated that wild-type amounts of ABA are not necessary for its accumulation during seed development. In all cases, LeGOLS-1 mRNA was most prevalent in the radicle tip. LeGOLS-1 mRNA accumulation was induced by dehydration but not by cold in germinating seeds, whereas both stresses induced LeGOLS-1 mRNA accumulation in seedling leaves. The physiological implications of LeGOLS-1 expression patterns in seeds and leaves are discussed in light of the hypothesized role of RFOs in plant stress tolerance.The raffinose family oligosaccharides (RFOs) are soluble galactosyl-Suc carbohydrates that constitute a significant component of phloem-transported sugars in some plants (Haritatos et al., 2000). Their accumulation in plants is associated with stressful environmental conditions such as cold, heat, or dehydration (Santarius, 1973; Santarius and Milde, 1977; Hinesley et al., 1992; Ashworth et al., 1993; Wiemken and Ineichen, 1993; Bachmann et al., 1994; Taji et al., 2002). The potential role of RFOs in stress tolerance has been intensively studied in seeds, particularly with respect to desiccation tolerance and longevity in the dehydrated state. RFOs are abundant in most mature desiccation-tolerant ("orthodox") seeds and are often rare or absent in "recalcitrant" seeds that cannot withstand desiccation (Lin and Huang, 1994; Sun et al., 1994). In seeds of many species, RFO accumulation coincides with the development of desiccation tolerance during seed maturation (Koster and Leopold, 1988; Leprince et al., 1993; Bewley and Black, 1994; Horbowicz and Obendorf, 1994; Black et al., 1996; Brenac et al., 1997a Brenac et al., , 1997b, and RFO content has been positively correlated with seed longevity in storage (Bernal-Lugo and Leopold, 1992; Horbowicz and Obendorf, 1994; Lin ...

ARGONAUTE proteins (AGOs) are known to be key components of the RNA silencing mechanism in eukaryotes that, among other functions, serves to protect against viral invaders. Higher plants encode at least 10 individual AGOs yet the role played by many in RNA silencing-related antiviral defense is largely unknown, except for reports that AGO1, AGO2, and AGO7 play an antiviral role in Arabidopsis (Arabidopsis thaliana). In the plant virus model host Nicotiana benthamiana, Tomato bushy stunt virus (TBSV) P19 suppressor mutants are very susceptible to RNA silencing. Here, we report that a N. benthamiana AGO (NbAGO) with similarity to Arabidopsis AGO2, is involved in antiviral defense against TBSV. The activity of this NbAGO2 is shown to be directly associated with anti-TBSV RNA silencing, while its inactivation does not influence silencing of transiently expressed transgenes. Thus, the role of NbAGO2 might be primarily for antiviral defense.

The SNF1/AMP-activated protein kinase subfamily plays central roles in metabolic and transcriptional responses to nutritional or environmental stresses. In yeast (Saccharomyces cerevisiae) and mammals, activating and anchoring subunits associate with and regulate the activity, substrate specificity, and cellular localization of the kinase subunit in response to changing nutrient sources or energy demands, and homologous SNF1-related kinase (SnRK1) proteins are present in plants. We isolated cDNAs corresponding to the kinase (LeSNF1), regulatory (LeSNF4), and localization (LeSIP1 and LeGAL83) subunits of the SnRK1 complex from tomato (Lycopersicon esculentum Mill.). LeSNF1 and LeSNF4 complemented yeast snf1 and snf4 mutants and physically interacted with each other and with LeSIP1 in a glucose-dependent manner in yeast two-hybrid assays. LeSNF4 mRNA became abundant at maximum dry weight accumulation during seed development and remained high when radicle protrusion was blocked by abscisic acid (ABA), water stress, far-red light, or dormancy, but was low or undetected in seeds that had completed germination or in gibberellin (GA)-deficient seeds stimulated to germinate by GA. In leaves, LeSNF4 was induced in response to ABA or dehydration. In contrast, LeSNF1 and LeGAL83 genes were essentially constitutively expressed in both seeds and leaves regardless of the developmental, hormonal, or environmental conditions. Regulation of LeSNF4 expression by ABA and GA provides a potential link between hormonal and sugar-sensing pathways controlling seed development, dormancy, and germination.

The specificity and sensitivity of polymerase chain reaction (PCR) primers developed for ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Liberibacter psyllaurous’ were evaluated in conventional and real-time PCR assays. All PCR primers were specific for ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ insomuch as they did not detect other prokaryotic plant pathogens that affect potato except for the putative pathogens associated with psyllid-yellows and haywire. Conventional PCR assays were capable of detecting 0.19 to 1.56 ng of total DNA per reaction, and real-time PCR was found capable of detecting 1.56 to 6.25 ng of total DNA per reaction, depending on the specific PCR primer set used. ‘Ca. Liberibacter’ species associated with zebra complex disease (ZC) was confirmed in plants affected by this disease throughout Texas from 2005 to 2008, in seed tubers produced in Wyoming in 2007, and in Colorado, Kansas, Nebraska, and Mexico in 2008. A multiplex PCR assay using ‘Ca. L. solanacearum’–specific primers and primers specific for the β-tubulin DNA regions from potato was developed, providing possible utility of the multiplex assay for ‘Ca. Liberibacter’ detection in different solanaceous plant species. Preliminary studies suggest silverleaf nightshade (Solanum elaeagnifolium), wolfberry (Lycium barbarum), black nightshade (S. ptychanthum), and jalapeno pepper (Capsicum annuum) as additional solanaceous hosts for the ZC-associated bacterium. The ‘Ca. Liberibacter’ species detected in all samples divided into two clusters sharing similarity of 99.8% in their partial 16S rRNA gene sequences and 99.3% in their partial intergenic spacer region (ISR)-23S rRNA gene sequences. Genetic variation in the 16S rDNA region consistently matched that of the ISR-23S rDNA region. In this partial 16S-ISR-23S rDNA region, there was a total of eight single nucleotide polymorphisms among ‘Ca. L. psyllaurous’ and ‘Ca. L. solanacearum’ “strains” investigated in this study. ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’ were shown to be very closely related bacteria, if not the same, by successful amplification using a combination of forward primer of ‘Ca. L. solanacearum’ and reverse primer of ‘Ca. L. psyllaurous’ in ZC-affected potato samples. This finding clarifies the current taxonomic status of ‘Ca. L. solanacearum’ and ‘Ca. L. psyllaurous’. The detection of ‘Ca. L. solanacearum’ from haywire-symptomatic potato samples demonstrates that this bacterium might also be associated with this disease.

RNA silencing is a common strategy shared by eukaryotic organisms to regulate gene expression, and also operates as a defense mechanism against invasive nucleic acids such as viral transcripts. The silencing pathway is quite sophisticated in higher eukaryotes but the distinct steps and nature of effector complexes vary between and even within species. To counteract this defense mechanism viruses have evolved the ability to encode proteins that suppress silencing to protect their genomes from degradation. This review focuses on our current understanding of how individual components of the plant RNA silencing mechanism are directed against viruses, and how in turn virus-encoded suppressors target one or more key events in the silencing cascade.

As seed dormancy is released within a seed population, both the rate and percentage of germination increase progressively with increasing dose of a dormancy-breaking treatment or condition. Population-based models can account for this behaviour on the basis of shifting response thresholds as dormancy is alleviated. In particular, hydrothermal time analysis of germination sensitivity to water potential (C) and temperature (T) can describe these features of seed behaviour. We used the hydrothermal time model to analyse the effects of dormancybreaking treatments on germination of dormant true (botanical) potato (Solanum tuberosum L.) seeds (TPS). After-ripening (378C and 4% seed moisture content) of TPS for 7 or 30 days partially or fully alleviated primary dormancy. The median base water potential required to prevent germination [C b (50)] decreased from 2 0.25 MPa in control seeds to 2 0.87 MPa and 2 1.83 MPa after 7 and 30 days of after-ripening, respectively. In contrast, the base temperature for germination (T b ) was relatively unaffected (0 -3.38C). Fluridone (50 mM), an inhibitor of abscisic acid (ABA) biosynthesis, also promoted germination of dormant TPS and lowered C b (50), indicating a role for de novo synthesis of ABA during dormancy maintenance. Moist chilling (3 days at 48C) or gibberellin (100 mM) alleviated secondary dormancy and lowered C b (50) values from 20.08 MPa to 2 0.36 and 20.87 MPa, respectively. The hydrothermal time model allows quantification of dormancy levels and explains why changes in germination speed and percentage are closely correlated during dormancy alleviation.

SummaryEndeavours to obtain elevated and prolonged levels of foreign gene expression in plants are often hampered by the onset of RNA silencing that negatively affects target gene expression. Plant virus-encoded suppressors of RNA silencing are useful tools for counteracting silencing but their wide applicability in transgenic plants is limited because their expression often causes harmful developmental effects. We hypothesized that a previously characterized tombusvirus P19 mutant (P19 ⁄ R43W), typified by reduced symptomatic effects while maintaining the ability to sequester short-interfering RNAs, could be used to suppress virus-induced RNA silencing without the concomitant developmental effects. To investigate this, transient expression in Nicotiana benthamiana was used to evaluate the ability of P19 ⁄ R43W to enhance heterologous gene expression. Although less potent than wt-P19, P19 ⁄ R43W was an effective suppressor when used to enhance protein expression from either a traditional T-DNA expression cassette or using the CPMV-HT expression system. Stable transformation of N. benthamiana yielded plants that expressed detectable levels of P19 ⁄ R43W that was functional as a suppressor. Transgenic co-expression of green fluorescent protein (GFP) and P19 ⁄ R43W also showed elevated accumulation of GFP compared with the levels found in the absence of a suppressor. In all cases, transgenic expression of P19 ⁄ R43W caused no or minimal morphological defects and plants produced normal-looking flowers and fertile seed. We conclude that the expression of P19 ⁄ R43W is developmentally harmless to plants while providing a suitable platform for transient or transgenic overexpression of value-added genes in plants with reduced hindrance by RNA silencing.