Environmental cues, especially temperature and light, have huge impacts on organ abscission. It has been reported in various plant species that high temperature accelerates reproductive organ abscission. In cotton, day temperatures above 40 °C can induce flower abscission. In soybean, flower abscission was found to increase with the elevated temperature treatment in three different soybean varieties, while no significant difference was found between control and cool temperature treatments. In addition, the light quality is also critical for organ abscission. Shading, as well as dark treatments, induced reproductive organ abscission in several plants. In pepper, shading treatment enhanced flower abscission in several cultivars. In apple, periods of darkness, shading, or cloudy weather have been showed to increase fruit abscission leading to early fruit drop. Nineteen days of shading treatment caused 98% of the fruit to abscise. In grape, five days of shading at bloom reduced the percentage of fruit set. However, the mechanisms of high temperature, or dark/low light-induced abscission and whether auxin is involved in these processes are still unknown. The DR5::GUS reporter system provides a visual indication of the activity of the auxin response in the auxin signal transduction pathway. We used tomatoes transformed with this reporter to investigate dynamics of the auxin response system during the different developmental stages and in response to environmental cues in pedicel AZ of tomato and to test the hypothesis that changes in the auxin response system are important in the regulation of abscission.
To investigate the dynamics of the auxin response system in pedicel during flower development,vertical plant growing we collected pedicels two days before anthesis , at anthesis, and 5 and 10 days post anthesis/pollination . We examined the distribution of the auxin response activity using the DR5::GUS reporter system. GUS activity was concentrated in the vascular tissues, with the majority of activity, particularly in the young flowers and those at anthesis, on the distal side of the abscission zone , a clear disjunction or ‘step’ in the auxin response activity at the abscission zone. At anthesis the GUS activity seen in the proximal zone of the younger flowers had disappeared, increasing the difference in the auxin response across the AZ. GUS staining in pedicels at 5 and 10 DPA was considerably enhanced, particularly in the abscission zone and in the vascular tissues of the proximal portion of the pedicel . To examine the relationship between these auxin response changes on the control of abscission, we removed flowers 2 DBA, at anthesis, and 5 DPA. All of the pedicels whose flowers had been removed at anthesis had abscised 12 h after flower removal, but there was no abscission of flowers from young and older flowers .Following flower removal, pedicels were treated at the distal end, or at the junction between the pedicel and the peduncle with lanolin containing 1 mM auxin. Four hours after the start of the experiment there was little obvious change in distribution or intensity of the GUS staining , indicating that the response system was not rapidly responsive to changes in auxin concentration.Eight hours after flower removal pedicels began to separate and most had abscised by 12 h. . We tested the changes in the distribution of the auxin response after flower removal using the DR5::GUS reporter system.
Four hours after flower removal GUS staining in the pedicels was similar to that in the controls . RT-PCR visualization of GUS expression in the tissues confirmed that there was little change in expression in the early stages of the abscission process . There was a perceptible decrease in the sharp ‘step’ in GUS staining across the abscission zone 8 h after flower removal, and even more in pedicels that had not yet abscised 12 h after flower removal, suggesting a reduction in auxin response adjacent to the abscission zone in the later stages of abscission. GUS expression in pedicels 16 h after flower removal was confined to the distal portion of the pedicel .Treatment with the aubegan to abscise and most had abscised by 12 h . We tested the changes in the xin transport inhibitor mimicked the effect of flower removal on pedicel abscission. Eight hours after the treatment pedicels distribution of the auxin response after NPA treatment using the DR5:: GUS reporter system. Four hours after flower removal GUS staining in the pedicels was similar to that in the controls . There was a perceptible decrease in the sharp ‘step’ in GUS staining across the abscission zone 8 h after flower removal , suggesting a reduction in auxin response adjacent to the abscission zone in the later stages of abscission.Treatment with 10 ppm ethylene accelerated flower abscission following flower removal , while pretreatment for 24 h with 1-methylcyclopropene completely inhibited abscission . GUS activity in ethylene- and 1-MCP-treated pedicels 4 and 8 h after flower removal showed similar patterns to those seen in the controls . The sharp reduction in activity at the abscission zone showed little change 4 h after flower removal, even in ethylene-treated pedicels that had already abscised but was somewhat reduced after 8 h both in ethylene-treated and in 1-MCP-treated pedicels .Auxin is considered to be a key hormone in the initiation of abscission; the accepted model suggests that reduced transport of auxin through the AZ results in sensitization of the AZ to ethylene, which induces the chain of hydrolytic and other processes that lead to cell separation.
In a previous study, we demonstrated that a knotted homeobox transcription factor, KD1, plays a role in abscission, apparently by modulating transport of auxin through the AZ. Silencing KD1 increased auxin in the abscission zone, and microarray analysis suggested that this was associated with the down regulation of auxin efflux transporters, particularly PIN9. The study also suggested that the change in auxin distribution across the abscission zone resulting from KD1 activity was associated with a change in the activity of the auxin response pathway, and the experiments reported here were designed to test that hypothesis.High activity was seen in the distal portion of the pedicel during flower opening, with a marked disjunction or ‘step’ on the distal side of the AZ. Following pollination, response activity increased substantially, particularly in the young fruit, the AZ, and in the proximal region of the pedicel. Our data did not support the hypothesis that changes in distribution or activity of the auxin response system play an important role in the regulation of abscission. Removing the flowers at anthesis, which induces pedicel abscission within 8 h had little effect on the distribution or activity of auxin response , particularly in the early hours after excision,vertical farming when the abscission process is initiated. The visual results from GUS staining of the pedicels are supported by RT-PCR analysis of the expression of GUS transcripts , which shows a marked ‘step’ in transcript abundance across the abscission zone, and a change in expression pattern only at 12 h after excision, when most pedicels have already abscised.The conclusion that a change in distribution or activity of the auxin response system plays no regulatory role in pedicel abscission is supported by our additional data. None of the other manipulations that affected the occurrence or timing of abscission had a marked effect on GUS staining. Placement of auxin distal or proximal to the abscission zone, treatment with NPA, treatment with ethylene, or 1-MCP , placing inflorescences in the dark, or at high temperature, all had significant and varied effects on abscission, but the distribution of auxin response system as indicated by GUS activity was remarkably stable. In contrast, the data presented here demonstrated that the early stages of abscission were associated with marked changes in the distribution and activity of genes involved in auxin transport. This is in agreement with our earlier results. Application of the auxin transport inhibitor, NPA, resulted in a marked reduction of expression of genes encoding enzymes involved in auxin transport . Particularly striking decreases were seen in the expression of PIN1, PIN6, PIN9, and AUX/LAX2 in the abscission zone itself. This general pattern was also seen following flower removal, although the reduction in expression of the PIN genes appeared to be less tissue specific .
These changes are consistent with the observations of Shi et al, who also found a substantial reduction in SlPIN1 expression following flower removal, and suggested that it might play a role in modulating the auxin content of the AZ. Silencing of SlPIN1 expression accelerated pedicel abscission by simultaneously increasing auxin accumulation in the ovary and decreasing the auxin levels in the AZ, suggesting that auxin transport modulates auxin balance to influence pedicel abscission. Interestingly, PIN2/5/10 were not expressed in pedicels . This is different from other reports that down regulation of PIN5 in the flower pedicel reduces intracellular auxin accumulation in the endoplasmic reticulum , which is expected to control auxin availability for auxin signaling/response in the nuclei of AZ cells. The exact regulatory roles of these auxin transporters in the induction of abscission need further investigation in the future. Auxin and abiotic stress work together affecting plant growth and development. In Arabidopsis, the shoot ward auxin transport can be inhibited by the reduction of PIN1/ 3 transcripts under low temperature and increased by the upregulation of PIN2 under high temperature. In addition, high temperature induces hypocotyl elongation by regulating PIF4-mediated auxin biosynthesis. Our data showed that both high temperature and darkness can accelerate abscission . However, the intensity and distribution of the auxin response were almost little affected by these substantial changes in environmental conditions . It is still unknown if auxin transport can affect the pedicel abscission in tomato under these environmental conditions and would require further investigation. Our results are consistent with a model that places the primary control of abscission on the concentration of auxin in the abscission zone. Concurrent changes in the relative rates of influx and efflux might plausibly result in marked changes in auxin concentration, triggering the sensitivity to ethylene that results in the onset of the abscission process. Our data indicate that at 4 h after flower removal, expression of genes encoding auxin efflux enzymes fell while the expression of genes encoding influx enzymes increased. We can imagine a scenario where the activity of KD1 is controlled by auxin transported from the flower. When auxin flow falls, KD1 might modulate the expression of genes involved in auxin influx and efflux, amplifying the effect of small changes in auxin flow, and resulting in a marked fall in auxin content of the abscission zone, triggering the changes that result in separation. In this scenario, the auxin response system is an important factor, but it functions as a reporter of auxin content, and does not rapidly change activity or distribution in response to changes in auxin supply from the flower.Tomato inflorescences were harvested at 10 AM from plants grown in the greenhouse at the University of California Davis. inflorescences with at least two newly opened flowers at anthesis , two days before anthesis and five days post anthesis were cut on the proximal side of the AZ and placed in vials, and held in a chamber into which humid air was continuously flown through. For testing abscission triggered by auxin depletion via flower removal, flowers were removed with a sharp razor blade by cutting on the distal side of the AZ, and abscission of the remaining pedicel from the peduncle was monitored at intervals. For testing abscission triggered by auxin transporter inhibitor, N-1- naphthylphthalamic acid , the inflorescences were placed in vials containing 10 ml of 25 μM NPA solution. Flowers were not removed for NPA treatments. Control inflorescences were placed in a vial containing a solution of the equivalent concentration of dimethyl sulfoxide. For testing temperature-dependent abscission, the inflorescences were harvested at the anthesis stage. The pedicels with/out flowers were placed in the testing chambers in temperature-controlled rooms with indicated temperatures. For dark-induced pedicel abscission, the inflorescences were harvested at the anthesis stage with/ out flower removal and placed in the chambers under the dark conditions at 20 °C. For testing ethylene-triggered pedicel abscission, the inflorescences were harvested at the anthesis stage with flower removal and placed in the ethylene chambers. All the experiments were carried out with at least three biological replicates.