Physiological and Genetic Characterization of End-of-Day Far-Red Light Response in Maize Seedlings

Developmental responses associated with end-of-day far-red light (EOD-FR) signaling were investigated in maize (Zea mays subspecies mays) seedlings. A survey of genetically diverse inbreds of temperate and tropical/semitropical origins, together with teosinte (Zea mays subspecies parviglumis) and a modern hybrid, revealed distinct elongation responses. A mesocotyl elongation response to the EOD-FR treatment was largely absent in the tropical/semitropical lines, but both hybrid and temperate inbred responses were of the same magnitude as in teosinte, suggesting that EOD-FR-mediated mesocotyl responses were not lost during the domestication or breeding process. The genetic architecture underlying seedling responses to EOD-FR was investigated using the intermated B73 × Mo17 mapping population. Among the different quantitative trait loci identified, two were consistently detected for elongation and responsiveness under EOD-FR, but none were associated with known light signaling loci. The central role of phytochromes in mediating EOD-FR responses was shown using a phytochromeB1 phytochromeB2 (phyB1 phyB2) mutant series. Unlike the coleoptile and first leaf sheath, EOD-FR-mediated elongation of the mesocotyl appears predominantly controlled by gibberellin. EOD-FR also reduced abscisic acid (ABA) levels in the mesocotyl for both the wild type and phyB1 phyB2 double mutants, suggesting a FR-mediated but PHYB-independent control of ABA accumulation. EOD-FR elongation responses were attenuated in both the wild type and phyB1 phyB2 double mutants when a chilling stress was applied during the dark period, concomitant with an increase in ABA levels. We present a model for the EOD-FR response that integrates light and hormonal control of seedling elongation.Plants utilize a complex network of photoreceptors to monitor the spectral quality, fluence, direction, and duration of light (Smith, 1995). These photosensory pigments include phytochromes that sense red light (R; 580–690 nm) and far-red light (FR; 690–800 nm) and the cryptochromes, phototropins, and zeitlupes for blue light (380–495 nm) and UV-A light (320–380 nm). The light reflected and transmitted by the vegetation creates a canopy characterized by reductions in both the R-to-FR ratio (R:FR) and the photosynthetically active radiation (400–700 nm). This light environment induces adaptive biochemical and morphological responses known as the shade avoidance syndrome (Smith and Whitelam, 1997). These responses can be induced early in development, before canopy closure, through FR reflected from adjacent neighbor plants (Ballare et al., 1990) or from low-lying weeds (Rajcan and Swanton, 2001), which can negatively impact yields in maize (Zea mays subspecies mays; Rajcan et al., 2004), even if only present early in the growing season (Liu et al., 2009).R:FR signals are transduced by the phytochrome family of photoreceptors (Franklin and Whitelam, 2007b). In rice (Oryza sativa) and sorghum (Sorghum bicolor), three genes constitute the phytochrome family: PhytochromeA (PhyA), PhytochromeB (PhyB), and PhytochromeC (PhyC). In maize, an ancient alloploidization has doubled the family size to six: PhyA1, PhyA2, PhyB1, PhyB2, PhyC1, and PhyC2 (Sheehan et al., 2004). Although many similarities are apparent between maize and Arabidopsis (Arabidopsis thaliana) light response, there are significant differences between members of the phytochrome gene family in copy number and selection pressures that have resulted in the divergence of phytochrome signaling networks (Sawers et al., 2005; Sheehan et al., 2007). Thus far, only three phytochrome mutants have been characterized in maize: elongated mesocotyl1 (elm1), phyB1, and phyB2. The elm1 mutant carries a mutation in phytochromobilin synthase, necessary for the biosynthesis of the chromophore common to all phytochromes (Sawers et al., 2004). The mutation severely reduces the total phytochrome pool, but the weak nature of the allele enables a partial responsiveness to R and FR (Markelz et al., 2003). At maturity, elm1 mutants have elongated internodes, pale green leaves, and flower early (Sawers et al., 2002). Mutations at phyB1 and phyB2 also impair light signal transduction. At maturity, both PHYB1 and PHYB2 contribute to plant height, stem diameter, and sheath-internode length, but PHYB2 predominates in the control of flowering (Sheehan et al., 2007). Like the sorghum and rice phyB mutants (Childs et al., 1997; Takano et al., 2005; Kebrom et al., 2010), both elm1 and phyB1 phyB2 double mutants constitutively display several traits associated with low R:FR response (Sawers et al., 2002; Markelz et al., 2003; Sheehan et al., 2007).In Arabidopsis, R/FR-mediated responses are developmentally complex and involve the PIF proteins (Duek and Fankhauser, 2005) and many hormones including auxins (Tao et al., 2008), ethylene (Khanna et al., 2007), jasmonate (Moreno et al., 2009), and GA (Djakovic-Petrovic et al., 2007). In particular, there is a direct interaction between PIF and DELLA proteins that govern phytochrome-mediated elongation (de Lucas et al., 2008; Feng et al., 2008; Lorrain et al., 2008). DELLA proteins also regulate FR inhibition of germination by abscisic acid (ABA; Piskurewicz et al., 2009), suggesting an interaction between the PIFs and ABA signaling. Complex cross talk between light and temperature has also been reported (Franklin, 2009). In Arabidopsis, colder temperatures can repress the early-flowering phenotype of the phyB mutant (Halliday et al., 2003). These studies suggest a complex interplay between light, hormone, and temperature to fine-tune the elongation response.The end-of-day far-red light (EOD-FR) treatment consists of a pulse of FR given at subjective dusk (Kasperbauer, 1971) and triggers a circadian clock-gated response (Salter et al., 2003). EOD-FR treatments result in a minimal pool of active Pfr during the dark period (Fankhauser and Casal, 2004), and plants submitted to daily treatments display similar developmental responses to those elicited by a continuous photoperiod with low R:FR (Smith, 1994). One of the key features that contributed to the discovery of the phytochromes is the photoreversibility of the response (Borthwick et al., 1952). These low-fluence responses (LFRs) are induced or repressed by alternating R and FR treatments (Mancinelli, 1994). The LFR nature of EOD-FR in maize was previously demonstrated in 5-d-old mesocotyl and coleoptile tissues (Gorton and Briggs, 1980). The EOD-FR treatment offers several advantages over growing plants in continuous low R:FR, including exposing plants to relatively brief treatment periods, thus potentially reducing genotype × environment effects. It also facilitates kinetic assays of phytochrome response, as treatments are limited to a single point in the diurnal cycle and can be delivered at any stage in plant development. Finally, as relatively low fluences of light are needed to saturate EOD-FR responses, large populations of seedlings can be screened without the need for large numbers of FR light-emitting diodes (LEDs) or sophisticated light chambers.Here, we have examined EOD-FR-mediated responses in maize and its closest relative, teosinte (Zea mays subspecies parviglumis). A survey of genetically diverse maize and teosinte accessions revealed extensive tissue-specific variations in mesocotyl, coleoptile, and first leaf sheath elongation. EOD-FR responses were greatly attenuated in tropical/semitropical (TS) accessions but present in teosinte, temperate inbreds, and a modern commercial hybrid, suggesting that the EOD-FR response is plastic in Z. mays. To investigate the genetic regulation underlying seedling responses to EOD-FR, we performed a quantitative trait locus (QTL) analysis using the intermated B73 × Mo17 (IBM) recombinant inbred population. We identified several QTLs that regulate mesocotyl and first leaf sheath response to EOD-FR and show that these QTLs mediate tissue-specific responses. The phyB1 phyB2 mutant series was also evaluated, indicating that the two PhyB paralogs are largely redundant in mediating the EOD-FR response. Pharmacological assays revealed a major role for GA in promoting mesocotyl, but not coleoptile or first leaf sheath, elongation in response to EOD-FR treatments. In contrast, EOD-FR reduced mesocotyl ABA levels. A chill treatment (10°C) applied during dark breaks attenuated EOD-FR elongation responses. Based on these observations, we discuss a model that integrates temperature, light, and hormonal inputs in the regulation of mesocotyl elongation.     

Abscisic acid catabolism in maize kernels in response to water deficit at early endosperm development

To further our understanding of the greater susceptibility of apical kernels in maize inflorescences to water stress, abscisic acid (ABA) catabolism activity was evaluated in developing kernels with chirally separated (+)-[(3)H]ABA. The predominant pathway of ABA catabolism was via 8'-hydroxylase to form phaseic acid, while conjugation to glucose was minor. In response to water deficit imposed on whole plants during kernel development, ABA accumulated to higher concentrations in apical than basal kernels, while both returned to control levels after rewatering. ABA catabolism activity per gram fresh weight increased about three-fold in response to water stress, but was about the same in apical and basal kernels on a fresh weight basis. ABA catabolism activity was three to four-fold higher in placenta than endosperm, and activity was higher in apical than basal kernels. In vitro incubation tests indicated that glucose did not affect ABA catabolism. We conclude that placenta tissue plays an important role in ABA catabolism, and together with ABA influx and compartmentation, determine the rate of ABA transport into endosperms.     

Inhibition of maize endosperm cell division and endoreduplication by exogenously applied abscisic acid

Abscisic acid (ABA) is thought to play a role in inhibiting or aborting kernel growth during water deficit. To test the responsiveness of early endosperm development to ABA concentrations, cylinders containing (±)ABA in a buffered agar medium were applied to the apical pericarp surface of kernels on intact, well‐watered maize ( Zea mays L. cv. Pioneer Brand 3925) plants from 5 to 11 days after pollination (DAP). Endosperm nuclei were analyzed by flow cytometry to assess effects on cell division and endoreduplication. ABA treatments of ≥ 100 µM substantially decreased endosperm cell numbers and fresh weight accumulation, but did not affect average cell size. ABA at ≥ 300 µM decreased the proportion of nuclei in the size classes ≥ 12C, indicating that the rate of transition to endoreduplication status was inhibited, and decreased the progressive advance from 12C to 24C to 48C, indicating that the rate of S‐phase cycling of endoreduplicating cells was inhibited. We conclude that cell division was more responsive to ABA concentrations than were endoreduplication or cell expansion growth.     

Gene and domain duplication in the chordate Otx gene family: insights from amphioxus Otx

We report the genomic organization and deduced protein sequence of a cephalochordate member of the Otx homeobox gene family (AmphiOtx) and show its probable single-copy state in the genome. We also present molecular phylogenetic analysis indicating that there was single ancestral Otx gene in the first chordates which was duplicated in the vertebrate lineage after it had split from the lineage leading to the cephalochordates. Duplication of a C-terminal protein domain has occurred specifically in the vertebrate lineage, strengthening the case for a single Otx gene in an ancestral chordate whose gene structure has been retained in an extant cephalochordate. Comparative analysis of protein sequences and published gene expression patterns suggest that the ancestral chordate Otx gene had roles in patterning the anterior mesendoderm and central nervous system. These roles were elaborated following Otx gene duplication in vertebrates, accompanied by regulatory and structural divergence, particularly of Otx1 descendant genes.      

Relationship between Coleoptile Elongation and Alcoholic Fermentation in Rice Exposed to Anoxia. II. Cultivar Differences

The relationship between coleoptile elongation and survivalvs. alcoholic fermentation of rice under anoxia is examinedusing eight cultivars differing in submergence tolerance. Anoxiawas imposed on either 1 or 4 d aerated seeds either by N₂ flushingsubmerged tissues or by incubating tissues in stagnant deoxygenatedagar at 0·1% w/v; the latter simulated the stagnant conditionsof waterlogged soil. Two cultivars that were most tolerant tosubmergence also had the greatest tolerance to anoxia, whilea submergence intolerant cultivar was also intolerant to anoxia. Coleoptile growth under anoxia was related to rates of ethanolsynthesis (R_E), however differences between growth during anoxiaand survival after anoxia indicated that post-anoxic injurymay also be important in rice seeds exposed to anoxia. The correlationbetween coleoptile growth and R_E measured on a tissue basisusing intact seeds was r² = 0·67 among six varietiesover 0-3 d anoxia. This correlation improved to about r² = 0·85using R_E of (embryos plus coleoptiles) over 0-3 d, or coleoptilesat 3 d after anoxia. Coleoptile growth of individual seeds wasusually poorly correlated to R_E in these cultivars at 2-3 dafter anoxia. When coleoptiles of similar lengths were obtainedfrom different cultivars using 4 d aerated seeds, there weredifferences in R_E and coleoptile growth which were related tocoleoptile growth during 3 or 5 d anoxia, either on a tissue(r² = 0·85) or a fresh weight basis (r² = 0·70-0·97respectively). Results are discussed in relation to factorswhich may limit ethanol synthesis in rice exposed to anoxiaand the importance of growth to the survival of seeds and matureplants during submergence in the natural environment.Copyright1994, 1999 Academic Press Anoxia, ethanol, alcoholic fermentation, Oryza sativa L., rice, submergence     

Characterization of a cellulose-binding, cellulase-containing complex in Clostridium thermocellum.

The isolation and biochemical characterization of the extracellular form of a cellulose-binding factor (CBF) from Clostridium thermocellum is described. The CBF was isolated from the culture supernatant by a two-step procedure which included affinity chromatography on cellulose and gel filtration on Sepharose 4B. The isolated CBF was homogeneous as determined by immunoelectrophoresis, polyacrylamide gel electrophoresis, gel filtration, and analytical ultracentrifugation analysis. The CBF was found to form a complex which exhibited a molecular weight estimated at 2.1 million. Electron microscopic analysis of negatively stained preparations of the isolated CBF revealed a particulate, multisubunit entity of complicated quaternary structure. The molecule appeared to be about 18 nm in size. Although urea failed to break the complex into its component parts, polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate resolved the CBF complex into 14 polypeptide bands. Immunoprecipitation experiments confirmed that these polypeptides indeed formed part of the same complex. Interestingly, by using the whole-cell immunization procedure described in the accompanying article (Bayer et al., J. Bacteriol., 156:818-827, 1983) only one CBF subunit (Mr = 210,000) was found to be antigenically active. By using a gel-overlay assay technique, at least eight of the remaining CBF-associated polypeptide components were shown to exhibit cellulolytic activity. The results are consistent with the contention that the CBF comprises a discrete, multisubunit complex or group of closely related complexes which exhibit separate antigenic and multiple cellulase activities in addition to the property of cellulose binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Partitioning of C-photosynthate, and long distance translocation of amino acids in preflowering and flowering, nodulated and nonnodulated soybeans

The influence of stage of development (preflowering versus flowering) in nodulated and nonnodulated soybeans (Glycine max [L.] Merr. cv. Wells) on partitioning of ¹⁴C into assimilates following exposure of a soybean leaf to ¹⁴CO₂ by both steady-state and pulse-labeling techniques was studied. Blades on the second fully expanded leaf from the stem apex were exposed to ¹⁴CO₂. Radioactive assimilates were extracted from source leaf blades, petioles, and stems (both the path up and path down from source leaf), were separated into neutral (sugars), basic (amino acids), and acidic (organic acids, sugar phosphates) fractions by ion exchange chromatography. The basic fraction was further resolved using thin layer chromatography and the percentage of radioactivity recovered in each amino acid was determined.     

Ice premelting during differential scanning calorimetry

Premelting at the surface of ice crystals is caused by factors such as temperature, radius of curvature, and solute composition. When polycrystalline ice samples are warmed from well below the equilibrium melting point, surface melting may begin at temperatures as low as -15 degrees C. However, it has been reported (. Biophys. J. 65:1853-1865) that when polycrystalline ice was warmed in a differential scanning calorimetry (DSC) pan, melting began at about -50 degrees C, this extreme behavior being attributed to short-range forces. We show that there is no driving force for such premelting, and that for pure water samples in DSC pans curvature effects will cause premelting typically at just a few degrees below the equilibrium melting point. We also show that the rate of warming affects the slope of the DSC baseline and that this might be incorrectly interpreted as an endotherm. The work has consequences for DSC operators who use water as a standard in systems where subfreezing runs are important.