Many DELLA mutants are constitutive repressors of the GA response and they have been made famous by the socalled “Green Revolution” which included wheat and rice plants that were dwarfed by their inability to respond to GA. Both D8 and D9 maize mutants do not respond to application of GA3 that causes an increase in height in normal siblings. Neither D8 nor D9 are as short as plants treated with PBZ, nor as short as GA biosynthetic mutants like d1, which suggests there are other GA receptor pathways in addition to D8 or D9 in maize.BLASTing the non-redundant maize genome with the FUN protein found partial homology to a region on chromosome 6, where chromosome 3 is known to have duplicates. This region does not translate into a continuous peptide chain, which could mean that duplicates of this gene are punished by selection. BLAST retrieved closely conserved homologues to FUN across the grasses. With PSI-BLAST it was possible to collect homologues of FUN throughout the Plant Kingdom, including Amborella trichopoda, Rosids, Asterids and nongrass monocots. The resulting tree assembled by MAFFT from these proteins fell into the same distribution as the APG IV phylogeny105, and is therefore assumed to be reliable . Alignment of the diverse family of FUN proteins revealed conserved regions , including one of the regions predicted by NucPred to be involved in nuclear localisation, which may be important to the function of FUN. Though BLASTing of ZmFUN only found one hit in the Brassicales , using the C. papaya gene as a BLAST query returned more Brassicale hits,vertical grow manufacturer allowing the retrieval of an Arabidopsis thaliana homologue. The Brassicales were found to have retained the FRWW, MRLM and KKR motifs, but not the GAKHIL motif . RNA-seq datasets published by Stelpflug et al. tracking different stages of maize development106 showed that the FUN transcript is highly expressed in the developing seed and endosperm.
The next highest peaks were: developing leaves, especially the base; immature and meiotic tassels; and the immature and pre-pollination cob. FUN transcript was detected in all samples examined in Stelpflug et al.’s dataset . Walley et al. collected transcript and protein from 23 developing maize tissues and mapped them to the v4 genome. FUN was also examined in this data set, where the highest transcript reads were found in developing leaves, and were much lower in mature leaves. Endosperm levels were still high, but not as extreme as seen in the dataset of Stelpflug et al. Unfortunately, immature tassels were not sampled, but strikingly, no FUN transcript was detected in mature pollen, unlike all other tissues sampled. Relatively high levels of FUN transcript were found in the female spikelet, as well as some in the silk . The proteomic data mapped to v4 did not return anything for FUN, but mapped to v3, high levels of protein were found in the endosperm and the developing leaves, but not the mature leaf, nor any other tissues sampled . No phosphorylated peptides were detected in any tissue sampled. Since these datasets were highly generalised, a dataset created specifically to query auricle development in maize was also used.A dataset created by Kong et al. compares developing B73 auricles with those of the inbre. B73 has what can be considered normal auricles, while the auricles of 986 are much smaller . Developing auricle tissue was collected at three stages and RNA-seq libraries were made. According to Kong et al.’s dataset, FUN is found at equal levels in the early auricle of both B73 and 986. At mid auricle development, both B73 and 986 drop, but 986 is lower than B73. Both have dropped to low levels by late auricle development . Thus, various RNA-seq datasets were used to give indications of where FUN localised at the macro level. Next, various software were used to predict where FUN localised at the sub-cellular level.The entire FUN protein amino acid sequence was entered into NucPred, an online tool for prediction of nuclear proteins as well as PSORT, which predicts sub-cellular localisation given a protein sequence.
The entire FUN protein sequence was entered into the PSIPRED prediction tool , which runs multiple protein folding and interaction predictions to come to a synthetic prediction for the structure and function of a given protein sequence. NucPred gave the FUN amino acid sequence a score of 0.98 on a scale of 0.1-1, with a score of 1 indicating certainty that the protein is nuclear . The k-NN Prediction provided by the online program PSORT lists FUN as 91.3% likely to be nuclear and 8.7% likely to mitochondrial. FFPred, part of the PSIPRED package, also agreed that FUN is likely to nuclear. FFPred also predicted that FUN is likely to be involved in DNA and RNA interactions, is likely to be involved in cytoskeletal and DNA binding, and has a high percentage of serines and glutmamic acid . DISOPRED, also part of the PSIPRED package, predicted that most of the FUN sequence can be defined as disordered, though the first 50 amino acids of FUN are not predicted to be disordered . In sum, FUN transcript is found in developing leaves and tassels, which fits with the phenotype of the mutant. FUN transcript is also found in developing ears, which is surprising since an ear phenotype was not observed. Higher levels of FUN during leaf development are associated with larger auricles. FUN is predicted to be a disordered protein that localises to the nucleus. Attempts were then made to validate these predictions. Y2H, using the FUN protein as bait, and a cDNA library of immature ears and tassels as prey, retrieved a list of 234 possible interactors. Using the A. thaliana homologues of these proteins in a GO term biological process analysis found enrichment for proteins involved in negative regulation organ, specifically flower, development; and proteins involved in negative regulation of nucleic acid metabolic processes . The A. thaliana homologues were enriched in transcription factor activity and hydrolase activity according to a GO term analysis based on molecular function , and most of these predicted interacting partners were nuclear or cytosol localised. The GRMZM numbers were also run through the GO term analysis prediction software provided by AgriGO and were found to be enriched in GTP and GTPase binding . A table of selected genes returned by the Y2H can be seen in Figure 5-7A. One such gene is ZmDWF1.
This brassinosteroid synthesis protein was found to have a synergistic interaction in double mutant analysis with fun . Another gene implicated in the brassinosteroids pathway was BSL1 that is known as an inhibitor of BRI1. The strongest confidence hit for the Y2H as an interactor for FUN was tubulin, butthis may be a false positive due to the high concentration of this protein in a cell.In order to test the idea that FUN is nuclear-localized, I carried out a transformation experiment using Nicotiana benthamiana. The entire FUN protein was fused to an N terminus YFP as described in Methods. The FUN-YFP fusion was found to localise to the nucleus in transformed N. benthamiana pavement cells. This result was observed in two separate transformations. Though not all nuclei in the samples were found to fluoresce under YFP excitation, this is likely due to imperfect transformation efficiency and is normal in this kind of experiment109. 25 nuclei expressing YFP were photographed and many more observed during the course of this experiment; YFP expression was not observed in any other sub-cellular regions. In order to confirm that the YFP expression was nuclear, the leaves were also examined under 405nm excitation. Since the leaves had been infiltrated with DAPI prior to examination,vertical grow marijuana system this caused the DNA to fluoresce. YFP fluorescence was shown to overlap with this DAPI fluorescence . As further confirmation, the sample was also examined under bright field and the YFP fluorescence was thus seen to overlap with clearly visible nuclei . This was observed at the microscope, as well as by overlapping micrographs using ImageJ. Close inspection of individual transformed nuclei revealed a nuclear speckle pattern . In order to make an antibody to the FUN protein, the purified protein has to be injected into a living animal and the antibody produced must then be purified. To this end, the third exon of FUN was amplified by primers 53xF/R and cloned into pENTR. Recombining with pDEST17 was unsuccessful. I hypothesised that FUN may be toxic, which would explain why the expression plasmid would not grow, though pENTR would grow, so bacteria were grown at lower temperatures. This was also unsuccessful, so a smaller fragment out of the exon was used, amplified from cDNA with primers An2F/R . This 600bp fragment was successfully cloned into pDEST17. Interestingly, the other fragments attempted that contained the conserved GAKHIL motif did not clone into pDEST17 under the conditions used, which could imply a toxicity of this domain, since all fragments that did not contain the GAKHIL motif were successfully cloned.The newly made plasmid pDEST17-An2F/R was then cloned into Rosetta cells and grown into a 100ml culture overnight. This culture was used to spike 1 litre of fresh LB. After 4 hours of growth at 37°C this culture reached 0.42 OD600nm and 200μl of 1M IPTG were added. This culture was then shaken at room temperature for 5 hours and spun at 8000 RCF for 15 minutes at 4°C. The resulting pellet was resuspended in 75ml of lysis buffer . This was spun at 12000 RCF for 15 minutes and the supernatant co-incubated with 2.5ml of 50% Ni++ beads in EtOH slurry.
The beads were spun and washed several times using wash buffer and were finally eluted using elution buffers at pH 4.4, 3.8 and 3twice each. Most protein was recovered from the second pH 4.4 and first 3.8 elutions as measured by nanodrop. These samples were then run on an SDS gel and the correct size band was observed for An2F/R samples, but nothing was observed for An1F/R. Thus the protein produced by plasmid pDEST17-An2F/R was purified. It was then resuspended in 6M urea by dialysis in a side-A-lyser cassette and sent to Cocalico for injection into guinea pigs. Test-bleeds and pre-bleeds were returned from the company. The pDEST17- An2F/R protein product was blotted onto nitrocellulose and incubated in 2ml of PBS with 2μl of test-bleed or 2μl of pre-bleed for 2 hours before washing and incubating in PBS and anti-Guinea Pig Alkaline Phosphotase fusion antibody. Finally this was incubated in NETN and NBT/BCIP to give an output of purple colour if the antibody is reacting to the protein . Later, second bleeds were returned by the company, and this dot-blot process was repeated, along with the sera from another antibody as a control . Thus, it was confirmed that the guinea pigs were producing appropriate antibody. In order to purify the terminal bleeds, a GST-tagged version of the protein was made. The pENTR-An2F/R was therefore combined with pDEST-15, transformed into Rosetta cells and induced and spun down as for the pDEST17- An2F/R purification step. This pellet was resuspended in NETN and put through a French press twice at ~1000lbs of pressure before 2x 20s of sonication to shear DNA. Triton X-100 was then added to 0.5%. This cell lysate was then spun at 12,000 RCF for 15mins and the supernatant was added to 2ml of 50% glutathione-sepharose beads equilibrated in 10ml NETN. After co-incubation during which the GST-tagged protein should bind to the beads, the beads are spun down and washed several times with 0.2M Borate pH8. The washed beads are then transferred to a column and cross-linked to the beads using DMP solution before washing with 0.1M glycine-Cl pH2.5 to remove non-covalently linked molecules and a final wash with 1xTBS . The terminal bleeds were thawed and 1/10 volume 10xTBS was added. This TBS-serum was then incubated in a column containing beads bound to GST for 30min at 4°C. Thus any antigens specific to GST that might be in the guinea pig sera would bind to these beads and the eluate from this column should be free of such contaminants. This eluate was incubated in the column described in the preceding paragraph and incubated at 4°C for 1 hour. Thus any antibodies specific to the FUN fragment should bind to these beads.