Oufti: an integrated software package for high‐accuracy,high‐throughput quantitative microscopy analysis

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re‐emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single‐cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand‐alone, open‐source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non‐diffraction‐limited fluorescence signals and is scalable for high‐throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post‐processing analysis, makes the software broadly accessible to users irrespective of their computational skills.     

RopGEF2 is involved in ABA‐suppression of seed germination and post‐germination growth of Arabidopsis

The involvement of Rho of Plants (ROP) GTPases in abscisic acid (ABA) signalling in Arabidopsis has been demonstrated in many studies. However, the roles of RopGEFs (Rop guanine nucleotide exchange factors), which modulate ROP activities in ABA signalling, are poorly understood. Here, we demonstrate that RopGEF2 may play a negative role in ABA‐suppressed seed germination and post‐germination growth. We show that disruption of RopGEF2 enhances sensitivity to exogenous ABA in seed germination assays and that RopGEF2pro‐GUS is mainly expressed in developing embryos and germinating seeds. Interestingly, YFP‐RopGEF2 is located in both the cytoplasmic region and in mitochondria. Notably, the PRONE2 (plant‐specific ROP nucleotide exchanger 2) domain of RopGEF2 is detected in mitochondria, whereas the N‐terminus of RopGEF2 is shown to be in the cytosol. After ABA treatment, degradation of RopGEF2 is triggered in the cytosol through the ubiquitin‐26S proteasome system. The binding of RopGEF2 to ROP2, ROP6 or ROP10, which has been demonstrated to be involved in ABA signalling, not only alters the localization of RopGEF2 but also enables RopGEF2 to escape degradation in the cell. Thus, in this study, we deduce a sophisticated mechanism of ABA‐mediated RopGEF2‐ROP signalling, which potentially implicates the inactivation of ROPs in responsiveness to ABA.     

胁迫时间与非毒性离子对重金属抑制拟南芥种子发芽及幼苗生长的影响

测定了Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺单一重金属胁迫对拟南芥种子发芽和幼苗生长的影响.结果表明,重金属对幼苗生长的毒性大于对种子发芽的毒性,以抑制种子发芽的IC₅₀为指标,4种重金属的毒性顺序为Hg²⁺＞Cd²⁺＞Pb²⁺/Cu²⁺,以幼苗生长为指标,则毒性顺序为:Cu²⁺＞Hg²⁺＞Cd²⁺/Pb²⁺,并随着胁迫时间延长,种子萌发率下降.此外,不同重金属在不同发芽时段对种子的毒性也不尽相同,Cd²⁺的毒性在种子吸水后的0～12 h大于12～24 h,而Hg²⁺毒性在12～24 h大于0～12 h,其中,种皮对减轻重金属毒性起着十分重要的作用.通过非毒性离子(Ca²⁺、Mg²⁺、K⁺、Na⁺)与重金属离子(Hg²⁺、Cd²⁺、Cu²⁺、Pb²⁺)交互作用对拟南芥种子发芽及幼苗生长效应的研究发现, mmol·L^-1的Ca²⁺、Mg²⁺、K⁺、Na⁺可以增强Hg²⁺对种子发芽的毒性,但对Cd²⁺的毒性却没有影响.对于幼苗来说,Ca²⁺、Mg²⁺、K⁺、Na⁺可以显著增强Hg²⁺的毒性,Ca²⁺可以缓解Cd²⁺的毒性,但却增加Cu²⁺的毒性,K⁺可以缓解Pb²⁺对幼苗的毒害作用.最后,本文对重金属的毒害机理进行了探讨.     

Arabidopsis acyl‐CoA‐binding protein ACBP1 participates in the regulation of seed germination and seedling development

A family of six genes encoding acyl‐CoA‐binding proteins (ACBPs), ACBP1–ACBP6, has been characterized in Arabidopsis thaliana. In this study, we demonstrate that ACBP1 promotes abscisic acid (ABA) signaling during germination and seedling development. ACBP1 was induced by ABA, and transgenic Arabidopsis ACBP1‐over‐expressors showed increased sensitivity to ABA during germination and seedling development, whereas the acbp1 mutant showed decreased ABA sensitivity during these processes. Subsequent RNA assays showed that ACBP1 over‐production in 12‐day‐old seedlings up‐regulated the expression of PHOSPHOLIPASE Dα1 (PLDα1) and three ABA/stress‐responsive genes: ABA‐RESPONSIVE ELEMENT BINDING PROTEIN1 (AREB1), RESPONSE TO DESICCATION29A (RD29A) and bHLH‐TRANSCRIPTION FACTOR MYC2 (MYC2). The expression of AREB1 and PLDα1 was suppressed in the acbp1 mutant in comparison with the wild type following ABA treatment. PLDα1 has been reported to promote ABA signal transduction by producing phosphatidic acid, an important lipid messenger in ABA signaling. Using lipid profiling, seeds and 12‐day‐old seedlings of ACBP1‐over‐expressing lines were shown to accumulate more phosphatidic acid after ABA treatment, in contrast to lower phosphatidic acid in the acbp1 mutant. Bimolecular fluorescence complementation assays indicated that ACBP1 interacts with PLDα1 at the plasma membrane. Their interaction was further confirmed by yeast two‐hybrid analysis. As recombinant ACBP1 binds phosphatidic acid and phosphatidylcholine, ACBP1 probably promotes PLDα1 action. Taken together, these results suggest that ACBP1 participates in ABA‐mediated seed germination and seedling development.     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.     

Arabidopsis thaliana seed germination and early seedling growth are inhibited by monoethanolamine salts of para-halogenated benzoic acids

Abstract

In spite of the simplicity of its molecules, the complex effects of benzoic acids on the regulation of plant growth are an increasingly attractive field of research to chemists and biologists. Halide substituted benzoic acids offer an excellent opportunity to explore the effect of electron withdrawing substituents (fluoro-, chloro-, bromo- and iodo-) on the response of plant growth stage. Under normal physiological conditions, benzoic acids are ionized molecules that exhibit low solubility in water. Monoethanolamine, a natural alkanolamine, was used to generate salts of monoethanolamine of halogenated para-substituted benzoic acids, new compounds with biological activity. This study reports on the biological effects of these substances at different concentrations on Arabidopsis thaliana seed germination and early seedling growth. Seed germination at 22°C, in a vertical position, under a photoperiod of 16 h light and 8 h darkness, was variable depending on the concentration of the compounds applied. Final germination percentages were similar for all treatments and control at 0.05 mM and 0.1 mM (exception p-Br BA and p-I MEASPBA). No germination occurred when seeds were treated with more than 0.5 mM. The results also revealed that the primary root length and the number of secondary roots are reduced in a concentration-dependent manner and also in relation to increasing atomic size of the substituents (F < Cl < Br < I). It is concluded that uptake rates of benzoic acid anions by roots decrease with a decrease in hydrophilic character of the anion and with an increase in molecular size.     

Effects of heavy metals on seed germination and early seedling growth of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis>

Seed is a developmental stage that is highly protective against external stresses in the plant life cycle. In this study, we analyzed toxicity of essential (Cu²⁺ and Zn²⁺) and non-essential heavy metals (Hg²⁺, Pb²⁺ and Cd²⁺) on seed germination and seedling growth in the model species Arabidopsis. Our results show that seedling growth is more sensitive to heavy metals (Hg²⁺, Pb²⁺, Cu²⁺ and Zn²⁺) in comparison to seed germination, while Cd²⁺ is the exception that inhibited both of these processes at similar concentrations. To examine if toxicity of heavy metals is altered developmentally during germination, we incubated seeds with Hg²⁺ or Cd²⁺ only for a restricted period during germination. Hg²⁺ displayed relatively strong toxicity at period II (12–24 h after imbibition), while Cd²⁺ was more effective to inhibit germination at period I (0–12 h after imbibition) rather than at period II. The observed differences are likely to be due in part to selective uptake of different ions by the intact seed, because isolated embryos (without seed coat and endosperm) are more sensitive to both Hg²⁺ and Cd²⁺ at period I. We assessed interactive toxicity between heavy metals and non-toxic cations, and found that Ca²⁺ was able to partially restore the inhibition of seedling growth by Pb²⁺ and Zn²⁺.     

Biphasic fluence-response curves for light induced germination of Arabidopsis thaliana seeds

Abstract With appropriate pretreatment of the seeds fluence-response curves for the induction of germination of Arabidopsis thaliana show two phases. A proportion of the population responds to very low fluence (VLFR), 10⁴–10^?2μmolm^?2 establishing 10^?4–10^?2% of the total phytochrome in the far-red absorbing form (Pfr) and a proportion of the population respond to low fluence (LFR), 1–1000 μmolm^?2, establishing 1–75% Pfr. The VLFR is nol normally seen because the pre-existing Pfr level satisfies the Pfr requirement or use of green safelight establishes more Pfr than necessary to saturate the VLFR. Endogenous Pfr was depicted by a 24 h 35°C treatment, presumably as a result of dark destruction and/or dark reversion to the red absorbing form of phytochrome (Pr), making it possible to visualize the VLFR. A short pulse of 35°C treatment in combination with an appropriate temperature regime is also able to sensitize a proportion of the seed population. The proportion of the population showing the VLFR is determined by the duration of the cold imbibition pretreatment as well as the duration of the 35°C treatment. Complex fluence-response curves were observed in which a proportion of the seeds being promoted in the VLFR range, were inhibited at higher fluences before being further promoted in the LFR range. This was particularly clear for seed batches being sensitized by a short 35°C treatment. The VLFR may be of significance in the natural environment, enabling seeds buried in the upper layer of the soil to germinate, where the fluence rate falls off sharply and the LFR is not satisfied. A model is presented to explain the two phases in the fluence-response curves.     

TiLIA: a software package for image analysis of firefly flash patterns

As flash signaling patterns of fireflies are species specific, signal‐pattern analysis is important for understanding this system of communication. Here, we present time‐lapse image analysis (TiLIA), a free open‐source software package for signal and flight pattern analyses of fireflies that uses video‐recorded image data. TiLIA enables flight path tracing of individual fireflies and provides frame‐by‐frame coordinates and light intensity data. As an example of TiLIA capabilities, we demonstrate flash pattern analysis of the fireflies Luciola cruciata and L. lateralis during courtship behavior.     

AtNG1 encodes a protein that is required for seed germination

The pentatricopeptide repeat (PPR) family of eukaryotic proteins has numerous members in plants and is important for plant development. In the present study, we cloned a novel PPR gene, designated AtNG1, and characterized the ng1 Arabidopsis mutant. Morphological and structural observation of an ng1 mutant revealed that its sexual reproduction and seed formation processes are essentially normal. The mature embryonic root of ng1 is fully developed and has a well-differentiated structure; however, ng1 seeds cannot germinate, even when supplied with supplemental hormones and nutrition. Further investigation showed that embryo expansion and root cell elongation fails to occur after water imbibitions. Transient gene expression analysis indicated that AtNG1 localizes in mitochondrion. This implies that the deficiency of mitochondrion function might be the reason for the failed seed germination. Thus, our finding confirmed that AtNG1 plays a critical role in the early process of seed germination.     

The effect of seed and rosette cold treatment on germination and flowering time in some Arabidopsis thaliana (Brassicaceae) ecotypes

The germination and flowering responses to cold treatment were investigated in 32 ecotypes of Arabidopsis thaliana. A month-long cold treatment at the seed stage decreased the time until flowering in all but one strain, whereas a 3-d cold treatment had little, or the opposing effect. A month-long cold treatment at the rosette stage also decreased the time until flowering, but was less effective than seed cold treatment. Seed and rosette cold treatments did not have an additive effect on time until flowering. Cold treatment usually increased the speed of germination, however no clear response patterns for the probability of germination were detected. These findings are discussed in relation to the life cycle of the plant.     

Morpho‐colorimetric analysis and seed germination of Brassica insularis Moris (Brassicaceae) populations

Brassica insularis is a perennial plant growing on both coastal and inland cliffs. Three seed lots from Sardinia were analysed using an image analysis system to detect differences in seed morphology, both within and among populations. Germination requirements at constant (5–25 °C) and alternating temperatures (25/10 °C), both in light and in darkness, were evaluated for all populations. In addition, the effect of a dry after‐ripening period (90 days at 25 °C) was also investigated. Morpho‐colorimetric analysis clearly identified seeds from different populations and discriminated three chromatic categories for seeds belonging to the Isola dei Cavoli coastal population, but not for the inland Masùa and the coastal Planu Sartu. Inter‐population variability was also observed in germination behaviour. B. insularis seeds germinated, with percentages up to 60%, in a wide range of temperatures (5–25 °C), and neither light nor dry after‐ripening affected final germination percentages. Moisture content measurements were made for seeds of each colour, but there were no particular differences among colours. Inter‐populational variability in germination behaviour may be a survival strategy for species growing under unpredictable environmental conditions, such as under Mediterranean climate, while heteromorphy may be due to independent evolutionary divergence processes of the Isola dei Cavoli population.