There were no help files to guide those who were new to the program. The program was written in FORTRAN to be run in a DOS environment, so naming files was limited to eight characters or less. If there were any input errors, then the program did not indicate what they were, just that there were errors. Data entry involved parsing the salts added to the solution into the individual metals and ligands, calculating their respective concentrations, and then entering these concentrations as the –log into a DAT input file. Some calculations may have taken several iterations, which involved having to save the file, run the calculation, examine the output file, then make appropriate corrections, input the data again, save the file, and make additional calculations. In discussing these issues with the authors of GEOCHEM-PC we were encouraged to build upon and improve the existing program, so that it would work in a Windows XP or Vista environment and would have increased power and function. Included in GEOCHEM-EZ are improvements which would be expected by modern users , while maintaining complete backward compatibility to the GEOCHEM-PC format. A customizable database of common salts has been included, which eliminates the need to parse and to calculate the concentration of each metal or ligand. In addition, berry pots the user is no longer limited to enter concentration as nM, µM, or mM, but can now enter the concentrations as g/L or mg/ L, provided the salts of interest are part of the salts database. These last two features will make data input more rapid and help in eliminating the most common user errors. The program does automatically check for errors in data entry, convergence, and case similarity.

The user can instantly preview input and output files and make necessary corrections , something that formerly involved having to save these files and run the calculations a second or third time. Output files may be customized by filtering the output tables prior to saving the file. Within the Help menu we have included a Unit Converter which can convert any salt in the database from g/L or mg/L to molar concentrations or vice versa. Shown below is the GEOCHEM-EZ interface. This example is for a basal Murashige –Skoog medium, with the salt, metal, and ligand concentrations entered in mg/L. Note that the entries are mostly salts that are contained within the salts database and are accessed via the drop down list on the left side. However, the user may still add individual metals or ligands, if that is preferred. This entry for the M-S medium represents a simple case . Note that there are two tabs open , representing two separate cases that are being run simultaneously. Many cases can be run at the same time, another feature that makes solution analyses more rapid.Why should one use this program to design experimental solutions? Many scientists have modified standard nutrient solutions for hydroponics-based research or for specific experimental treatments without having analyzed these solutions to see whether any precipitation or solids may form because of the changes that they made to the solution composition. Geochem-EZ can help predict potential problems in experimental media. This program can also be used to design sensible chelate buffer systems or to calculate the concentration of a particular ion needed to provide a constant ionic activity. It is also a good way to know whether there is sufficient free activity of important nutrients in the solution of interest.

Often there is the assumption that if the nutrient is part of the solution, then it is readily available to the plant. This is not necessarily true. Interaction with other ions, pH effects, complexation, and precipitation may occur, reducing the free activity of the ion of interest. CO plays a role in the photoperiodic response in several grass species, such as rice , sorghum , and barley . However, these species also possess an additional photoperiod pathway that is not present in Arabidopsis, in which the PHOTOPERIOD1 gene plays a central role. PPD1 encodes a member of the PSEUDO RESPONSE REGULATOR protein family and is homologous to the Arabidopsis circadian clock genes PRR3 and PRR7 . The duplication that originated PRR3 and PRR7 in Arabidopsis and PRR37 and PRR73 in the grasses are independent, and therefore their sub-functionalization is independent . In Arabidopsis, PRR3 and PRR7 encode components of the circadian clock, and their disruption alters the expression of other clock genes . By contrast, variation in PPD1/ PRR37 in the grasses has no impact on the periodicity of the circadian response . These results suggest that after its duplication in the grass lineage, PPD1 evolved as a photoperiod gene that functions as an output of the circadian clock. Most natural variants in the photoperiodic response in wheat are associated with deletions in the promoters of PPD-A1 or PPD-D1 or with differences in PPD-B1 copy number . The promoter deletions in the PpdA1a or Ppd-D1a alleles are associated with the misexpression of PPD1 during the night, the induction of FT1, and the acceleration of flowering under SD . Plants carrying these alleles still flower earlier under LD than under SD and, therefore, will be referred to as “reduced photoperiodic response” alleles . The acceleration of flowering by PPD1 requires its transcriptional activation by light, which is mediated by two members of the phytochrome family, PHYB and PHYC .

The phytochromes absorb light maximally in the red and far-red spectrum and exist as two interchangeable isoforms, the inactive R light absorbing Pr form synthesized in the cytoplasm and the active FR light absorbing Pfr form that is translocated to the nucleus . Upon arriving in the nucleus, Pfr phytochromes interact with bHLH proteins known as PHYTOCHROME-INTERACTING FACTORS , which initiates a cascade of light regulated signaling pathways . During darkness and upon exposure to FR light, Pfr phytochromes revert to the inactive Pr form. Despite the molecular characterization of some of the components of the PPD1-dependent flowering pathway in wheat, there are still large gaps in our knowledge of the mechanisms involved in the light regulation of PPD1 and FT1 and in the perception of photoperiodic differences. In this study, we characterized the response of wheat when exposed to short pulses of light during the long nights of SD photoperiods, which are referred to as night-breaks or NBs hereafter. NB experiments have the advantage of modifying photoperiods without changing the total hours of light received by the plant. NBs cause significant delays in flowering when applied to SD plants grown under SD.These observations demonstrate that the duration of the night is critical to regulate flowering time in many SD plants and that the NB response can be characterized as a transient period of sensitivity to light that inhibits flowering. In rice, a single NB was sufficient to inhibit flowering in SD via the PHYB-mediated transcriptional repression of Hd3a . These observations are consistent with the external coincidence model of flowering, according to which flowering is induced when external light and internal oscillating circadian signals coincide . In this study, we show that NBs accelerate flowering in wheat plants grown under SD and that the response is strongest in the middle of the night. Using ppd1-null mutants, we demonstrate that this response is mediated by PPD1. We also show that although PPD1 transcription is rapidly induced within 1 h of exposure to a single NB, multiple NBs are required for induction of FT1 to high levels and for early flowering. Finally, we show that the magnitude of PPD1 induction in response to NBs increases in accordance with the length of darkness preceding the light signal and that this induction is dependent on active protein synthesis during darkness.When the long nights of SD were interrupted by 1 h pulses of white light at different points of the night , flowering of the Kronos-PS plants was accelerated . The timing of the NB had a strong effect on heading date, with a maximum acceleration when the NB was applied in the middle of the night . Under these conditions, plants headed just 7 d later than those grown in a LD photoperiod . NBs applied either earlier or later than this point had a weaker effect on heading date, although among plants exposed to NBs after 6, 8, or 10 h of darkness,hydroponic grow system heading date was not significantly different . NBs of 15 min given after 8 h of darkness were equally effective in accelerating flowering as 1 h NBs applied at the same time . To characterize the transcriptional responses associated with accelerated flowering in NB, we compared the expression levels of selected flowering time genes in 6-week-old plants grown since germination under NBmax conditions with those maintained in a SD photoperiod. Because allelic variation at the PPD1 loci can affect the expression of each homeolog separately, we measured PPD-A1 and PPD-B1 transcript levels using homeolog specific assays. For all other targets, quantitative reverse transcription -PCR assays that amplify both A and B homeologs were used . In SD-grown Kronos-PS plants, PPD-A1 and PPD-B1 expression levels remained low throughout the night, and FT1 transcripts were not detected at any of the analyzed time points.

In plants grown in NB conditions from germination, PPD-A1 transcript levels doubled in response to NB, but this homeolog was expressed at very low levels in all assayed time points . By contrast, PPD-B1 transcript levels were approximately 20-fold higher than PPD-A1 before NB and 26-fold higher after NB , suggesting that the PPD-B1 homeolog contributes the majority of PPD1 transcripts in photoperiod-sensitive tetraploid wheat. This result is consistent with a previous study in the hexaploid wheat variety Paragon, where PPD-B1 accounted for 90% of all PPD1 transcripts . PPD-B1 expression was significantly higher in NB than in SD conditions at all time points and was rapidly upregulated by NB, peaking between 1 h and 3 h after the start of the NB . FT1 transcript levels were significantly higher in NB conditions than in SD and showed increased expression 5 h after the start of the NB . Even before exposure to NB, FT1 transcript levels were significantly higher in plants grown under NB since germination than in those grown under SD . FLOWERING LOCUS T2 and VERNALIZATION1 expression levels were also elevated in plants grown in NB, while FLOWERING LOCUS T3 expression was reduced in comparison to SD-grown plants . These results show that the transcriptional regulation of these flowering time genes in NB is similar to their regulation in LD photoperiods .Phytochromes are activated and inactivated following exposure to R and FR light, respectively, so we tested the effects of FR light treatment on the NB response. Kronos-PS plants were grown under two different conditions from germination. In one chamber, plants were exposed to a 1 h NB after 8 h of darkness, and in the other chamber, plants were exposed to the same conditions except that the 1 h NB was followed by a 30 min pulse of FR light. Plants exposed to FR light exhibited a delay of 8.9 d in heading date when compared to control plants, but the difference was not significant . One possible reason for the mild effect of this FR treatment on heading date could be that the exposure to 1 h of white light was sufficient for the irreversible activation of downstream genes or proteins in the flowering induction pathway before the FR light inactivation of the phytochromes. To test this possibility, we applied the NBs as 15 1-min pulses of white light intercalated either with 15 1-min periods of darkness or 15 1-min pulses of FR light . Application of the NB using this protocol was less effective in accelerating heading than when the NB was given as a 1 h block of white light, but the FR treatment had a proportionally larger effect and significantly delayed heading date . At the transcriptional level, PPD-B1 expression was significantly reduced only by the pulsed FR treatment . These results suggest that despite the absolute requirement of PHYB and PHYC function for the NB response, the FR light conditions used in these experiments were not sufficient to abolish the NB response completely.