In order to avoid this issue, the present study examined the effects of fruit load in A. thaliana. Although an annual species, this plant produces many more fruits than alternate bearing trees, and has the potential to exacerbate the fruit load experienced by the inflorescence meristem. To identify how the fruit load changes expression patterns, surgically dissected IM apices were collected after producing more than 30 fruits, and were profiled with ATH1 microarrays. This analysis found relatively few differentially expressed genes, though the overall pattern was consistent with carbohydrate starvation and clearly supports the competition model. No evidence of flower inhibition was found, and instead flower promoting genes were strongly up regulated, which is inconsistent with the inhibitor hypothesis. The fruit dominance model was only weakly supported by the present data, though this may be related to an atypical dominance mechanism in A.thaliana. Further analysis of IM growth patterns supported the existence of a determinate growth pattern,growing raspberries in pots which displayed both fruit load dependent and independent effects. The arrest of IM activity was correlated to rosette leaf senescence, and eventually terminated through a localized senescence mechanism.
In order to observe how fruit load affects meristem activity, a microarray experiment was performed to correlate gene expression patterns with the three alternate bearing hypothesis. However, because microarrays only measure the relative concentration s between different transcripts, this procedure cannot directly identify the cause of the observed levels. The input from mRNA biosynthesis is differentially regulated than the catabolism processes that reduce the overall transcript pool, and both forms of regulation are in turn affected by changes in any of the multitude proteins that control them. In order to obtain this regulatory information, it would normally be appropriate to perform additional microarray experiments in the presence of chemical inhibitors to block key enzymatic steps, and to validate the actual expression levels in a subset of genes with RT-PCR or qPCR methods. Unfortunately, due to the circumstances of Dr. Smith’s departure, none of this work was performed as expected. Consequently, the data presented below is best interpreted as a preliminary analysis, providing a number of hypothesis that might be investigated in future studies. To begin, meristem tissues were surgically dissected from A. thaliana 1° inflorescences using plants grown in both long day conditions , and from plants grown in continuous light . Total RNA was extracted from each replicate, and then hybridized to five individual Affymetrix GeneChip ATH1 microarrays . An initial analysis revealed less than 100 differences between the two photoperiod treatments, and no clear patterns were found in any cellular functions, gene families, or metabolic pathways. In the absence of any significant differences between the two photoperiods, the data sets were combined, providing a total of five biological replicates for the high fruit load treatment.
Unfortunately, due to the abrupt end of my work in this lab, control tissues from de-fruited plants could not be collected as expected. Instead, a previously published microarray data set was chosen to replace the missing controls, providing 3 biological replicates. From the combined 5 x 3 array analysis, 15,473 genes were detected at least once, and 7337 were detected in all eight arrays. This list was further filtered to remove genes that had Bayesian probability scores below 1.5, and filtered to remove genes than had an adjusted p-values greater than 0.05. To focus on those that were most significantly affected by fruit load, only genes with logFC values above +2 or below-2 were selected for further analysis, providing a list of just 512 genes. Initial examination of functional annotations however, revealed a large number of genes known to be exclusively expressed in the pollen, most of which had elevated transcript levels in the high fruit load treatment. Curiously, this pollen signal was shared with a previous meristem profiling experiment, where it seemed equally anomalous This prompted a re-evaluation of the tissue collection procedure used in the present study, which revealed a highly probable route of pollen contamination that had previously been overlooked. This finding strongly suggests that the pollen-specific genes detected in both studies are false positives. To remove this source of bias, the expression pattern of all 512 genes was scored using the eFP browser. This found 66 genes that were exclusively expressed in the pollen, and another 33 that were specific to green embryos, and are also likely products of contamination. In contrast, 17 genes were expressed largely in immature flower buds, but unlike the pollen signal, these were often had severely reduced transcript levels. Instead of contamination, the reduction of flower bud specific transcripts most likely reflects a tissue collection bias The tissue for the control arrays removed flower buds around the SAM older than stage 6, while the present study removed flower buds older than about stage 3 . All 117 atypical genes were subtracted from the total, leaving a final list of 389 differentially expressed genes . Of these genes, 103 had reduced transcript levels, and 286 had increased transcript levels. Mapping of functional annotations within this data set revealed no clear trends, but instead found a widespread pattern where most functional pathways were represented by at least one or two genes.
Although coverage is sparse, in many cases it was possible to estimate the direction of metabolic flux and the larger impacts on cell physiology. When compared to SAM tissue collected under low fruit load conditions, the high fruit load treatment correlated with reduced transcript levels for several cell wall components, including the glycoproteins PRP2, PRP4, and the putative leucine-rich protein At4g18670. The existing glycoproteins may also be degraded, as the xyloglucan hydrolase TCH4 transcripts were elevated. These observations also parallel the findings for lipids, where several biosynthetic genes had reduced transcript levels , whereas one lipid catabolism gene showed an increased transcript level . The reduction of two potassium channel transcripts, KUP7 and KEA2, might also suggest that the plasma membrane and chloroplast envelope have a reduced capacity to maintain normal charge separation.For the high fruit load treatment the meristem tissue was collected near the time of apical arrest, after which the meristem tissues often became visibly dehydrated. To see if the early symptoms of dehydration were already present when the tissue was collected, the data was compared to young meristems in the absence of fruit load. This revealed a reduction in the transcripts of CER1, a gene involved in wax biosynthesis,plastic plant pot suggesting a loss of the waxy cuticle. Desiccation stress responses are indicated by the elevated transcript level of the abscisic acid biosynthesis gene NCED3. Several other abscisic acid responsive genes had elevated transcripts, including HVA22J, TSPO, HB5, and so did genes known to be up regulated by drought stress, such as HIS1-3, ERD14, and RD20. Most intriguingly, the high transcript levels of METACASPASE3suggests the existence of a programmed cell death response in the apical tissues. Although the exact role of MC3 is not well understood in plants, in animal models PCD is typically activated following the rupture of the mitochondria, flooding the cytoplasm with free radicals that then triggers a proteolytic cascade regulated by CASPASE enzymes. Depending on the tissue type and the nature of the stimulus, the destruction of the cell can follow one of three recognized patterns: necrosis, where the cell is rapidly destroyed by physical damage to its membranes, apotosis, where the cell breaks apart into small fragments that are eventually cleaned up by macrophages in the bloodstream, and autolysis, were significant portions of the cytoplasm and nuclei are enclosed in lytic vesicles and digested. Apotosis is further correlated with DNA laddering produced by the fragmentation of nucleosomes, while necrosis and autolysis tend to produce high molecular weight smears. In plant tissues, the mitochondria have been found to play a similar role in autolysis pathways, and this is supported by the present study, which found that two carbonic anhydrase transcripts, BCA3 and BCA6, were elevated under high fruit load treatment. The proteins produced by these two genes are localized to thechloroplasts and mitochondria, and this pattern suggests an increase in the concentration of dissolved bicarbonate ion. However, no other freeradical responses were detected in the current data, so the significance of this observation is unclear. In order to discriminate between the three modes of cell death, multiple attempts to observe the characteristic DNA laddering of apotosis were performed. Surprisingly, this found traces of DNA laddering in actively growing meristems, but not in older quiescent meristems, which instead produced high molecular weight smears . The necrotic mechanism might be indicated by the previous predictions for a weak cell wall, and by the presence of senescent pathways, though it is unlikely in the absence of any obvious sources of physical damage. Autophagy in contrast, is supported by the high transcript levels of AUTOPHAGY 8H , which may also have a role in ubiquitin-related protein degradation.
Under high fruit load conditions three catabolism genes were found to have higher transcript levels, which suggests that nitrogen is released as a part of protein catabolism. One is At4g33150, which is involved in lysine and ɑ-ketoglutarate hydrolysis, and the other is At5g18860, involved in nucleoside hydrolysis. The third gene, the methionine gamma-lyase At1g64660, more specifically suggests that nitrogen is released as ammonia. This is at odds however, with the increased transcript levels of the glutamine synthetase GLN1-4 and the nitrate importer NRT1.2, both of which suggest nitrogen shortages. The elevated transcripts of AGL44 is more difficult to interpret, because although AGL44 is annotated as a nitrate responsive gene, this gene does not always respond to nitrogen starvation.Compared to the absence of fruit load, meristems treated with high fruit load appear to convert their starch reserves into sucrose and exported the sugar out of the SAM. This is supported by the increased transcript levels of two putative Trehalose 6-Phosphate Phosphase genes which suggests that less starch biosynthesis occurs, and this correlates well to the increased transcript levels of the starch degrading enzyme AMY1. Although there are no detected genes for glycolysis or the TCA cycle, the simultaneous increased transcription of the sugar importers SUC2and ATPLT5suggests that the meristem cells are experiencing a carbohydrate shortage. Calcium signaling pathways are over-represented in the data set, comprising 48% of all detected signaling components, most of which show increased transcript levels under high fruit load conditions. Those with annotated functions suggest a role in wound, phosphate starvation and/or desiccation responses. These functions are also shared with the WNK4 kinase, which strongly implies that these signaling components have role in the senescent pathways described above.Mutants of FZL are known to produce irregular grana structure, while the over expression of ELIP2 reduces chlorophyll content. These findings mirror the reduction of FZL transcripts and the increase of ELIP2 transcripts detected in the present study, and suggests that chloroplast function has been compromised. Most chromatin binding proteins had reduced transcript levels under high fruit load, such as FAS1, ATX1, DMT7, and BRAHMA. Mutants in FAS1 are known to reduce heterochromatin, so the reduction of FAS1 here might lead to an increase of euchromatin. This is also supported by the reduced transcript levels of the histone methyltransferase ATX1and the DNA methyltransferases , which would likely increase the amount of euchromatin simply by shifting the balance toward acetylated histones, and by making it harder to maintain silenced DNA though cell division. However the reduction of the chromatin remodeling factor BRAHMA suggests that transcription rates in general might be reduced, despite the possible increase in exposed DNA. There is also evidence of DNA damage, as two nucleases had elevated transcript levels, while a loss of double-strand break repair is suggested by the reduction of HDG12 transcripts. Preliminary analysis of the microarray data indicated a strong senescent response, which was unexpected given that several steps had been taken in order to avoid senescent tissue. Reasoning that the tissue collection procedure had relied on an inadequate predictor of senescence, an attempt was made to identify better markers for future use. This was done by measuring leaf and bud growth rates every two days over entire lifespan of the plant, then searching for strong correlations that occurred at least 3-4 days before the symptoms of senescence became visible. Under the long day conditions, Col-0 plants were found to have a population of immature flower buds that varied over time, reaching a maximum at the first flower and then declining briefly before leveling off at about 10 buds .