Treatment with ABA promotes the large increase in ET production and hastens petal wilting in carnation flowers. Pretreatments with silver thiosulfate , a chemical that inhibits the perception of ET by the ET receptor, completely prevents the increase in ABA levels. A negative relationship was observed between the level of CKs and petal senescence in petunia and carnation. In rose, the increase of CK content antagonized petal senescence prompted by ET. Applying CKs delayed petunia petal senescence. Auxin also plays a role in ET-sensitive petal senescence. Application of auxin prompted ET production and petal wilting in cut carnation flowers. In addition, 2,4-dichlorophenoxyacetic acid , a synthetic auxin, induced the expression of 1-aminocyclopropane-1-carboxylic acid synthase genes in petals. In most research, these hormones are used as exogenous regulators to observe ET sensitivity and floral longevity in ET-sensitive species. Although the enrichment of ‘response to 1-aminocyclopropase-1- carboxylic acid and auxin stimulus’ was observed 12 h after pollination in the petals of petunia, the differential expression patterns of genes related to these hormones in petal senescence is unclear. Petal senescence is regulated by transcription factors . On one hand, ethylene-insensitve-like and ethylene response factors are correlated with the ET response signaling pathway. EIL3, a homolog of ETinsensitive 3 in carnation, is a pivotal switch of ET induced gene expression. DAFSAG9, which is homologous to ERF2, was significantly upregulated in senescing daffodil petals. On the other hand, a large group of other TFs, such as B-box zinc finger, bHLH DNA-binding, homeodomain-like , MADS-box, MYB, and NAC, display differential expression when ET-insensitivity is induced in the etr1-1 transgenic petunia. More than 20 members from the ERF, NAC, bZIP, HD-Zip, and WRKY TF families showed differential expression in petals at the early stage of pollination-induced senescence in petunia.

In addition, NAC, Aux/IAA, MYB, bZIP, and MADS-box are differentially expressed during carnation petal senescence. These studies indicate that these TFs play regulating roles in ET-dependent petal senescence. However, the biological functions of these TFs are largely unknown. High-throughput gene expression analysis using messenger RNA sequencing represents the most powerful tool to elucidate the underlying regulatory mechanism of corolla senescence. Recently, pollination- and ET-induced corolla senescence in petunia has been studied through RNA-Seq analysis16,20, however, the regulatory mechanisms that govern the onset of natural corolla senescence from opening to wilting in petunia is unclear. Therefore, identifying the dynamic processes and regulatory factors in transcription is a crucial step in determining the master switches in corolla senescence. We employed RNA-seq technology to investigate the global and chronological sequence of transcriptional events during the initial corolla senescence in petunia. Furthermore, virus-induced gene silencing system was used to dissect biological functions of potential regulatory genes such as TFs. Our data suggest that hormonal interactions between auxin and ET may play a critical role in the regulation of onset of corolla senescence in petunia.One microgram of total RNA was reverse-transcribed using PrimeScript RT reagent with gDNA Eraser Kit , according to the manufacturer’s instructions. Specific primers were designed by the Primer 3 program and listed in Supplementary Table S1. Amplifications were performed in an Applied Bio-systems 7300 system . Melting curve analysis was performed and the absence of non-specific products and primer dimers were verified. For data analysis, average threshold cycle values were calculated for each gene of interest, on the basis of three independent biological samples and were normalized and used to calculate relative transcript levels as described elsewhere. 26S ribosomal RNA was used as an internal standard for normalization.ET emission was monitored using a laser-based ET detector and a gas handling system as described previously. Briefly, flowers collected at D0 were placed into 70 ml sealed glass vials.

The air was passed through a platinum-based catalyzer before entering the cuvettes in order to remove external ET and other hydrocarbons. A scrubber with KOH and CaCl2 was used to reduce the CO2 and the water content in the gas flow. ET emission was monitored and recorded in real time. Three biological replicates of every flowering stage were performed. Each experiment was repeated three times.To measure longevity of intact flowers, white flowers from pTRV/CHS-TFs inoculated petunia ‘Primetime Blue’ plants were tagged at D0. The time when the corollas wilted and the edges collapsed was recorded. At least 20 flowers of three plants from each of the three independent biological replicates were monitored. Purple flowers from water-inoculated wild-type and white flowers from pTRV/CHS-silenced plants were used as controls. Statistical analyses were performed using the SPSS package . One-way analysis of variance was performed for experiments with one independent variable. Duncan’s test was used as the post hoc test if significant differences were found.Flowers that were fully opened but anthers not yet dehisced were marked as D0. The corollas continued to expand for 2 days. Visible senescence symptoms, such as curving of the corolla edges,nft channels were observed at an average of 4 days. Corolla wilting was found at about 7–8 days . We measured ET production using a real-time ET detection system, EDT-300. An increase and decrease of ET emission was detected during D2–D7 stage. The level spiked around D4, reaching the maximum level at 5.5 days, and then decreasing sharply .In order to determine the alteration in gene expression during corolla senescence, we generated cDNA libraries composed of the samples collected from four developmental stages with two biological replicates. RNA sequencing of these libraries produced 49,421,030, 52,985,600, 47,813,446, and 56,552,704 clean reads at D0, D2, D4, and D7, respectively . The sequences were mapped to the P. axillaris reference genome for annotation of all unigenes. The mapping rate was over 93% for samples of each stage . Differential expression analysis was conducted by comparing four different developmental stages. Analysis on all four stages generated 5167 unigenes that were significantly differentially expressed across these stages. The number of DEGs was decreased from 4626 between D0 and D2, to 1116 between D2 and D4, and to 327 between D4 and D7 . DEGs were clustered to generate expression patterns based on time series using the STEM software. Cluster analysis of the data from four time points generated 26 clusters, including down regulated genes in clusters 0 through 12 and upregulated genes in clusters 13 through 25 . A few clusters displayed a more complex pattern. For instance, clusters 2, 5, 7, 8, and 11 showed an initial decrease followed by upregulation. However, clusters 14 and 17 exhibited an initial increase followed by a decline . In addition, the down regulated clusters 3 and 4 and the upregulated clusters 15, 16, 21, 24, and 25 were statistically significant .In order to identify up and down regulated GO at each selected time point, seven gene clusters exhibiting either significantly decreased or increased expression were further analyzed using Cytoscape software with its GO enrichment tool BiNGO. At the transition from D0 to D2, the metabolic processes of major macro-nutrients including ‘carbohydrates, lipids, aromatic amino acids, and nitrogen compounds’ were down regulated . In addition, ‘cell wall organization and bio-genesis’, ‘Sadenosylmethionine biosynthesis’, and ‘negative regulation of transcription, DNA-dependent’ and ‘RNA metabolism’ were down regulated . However, ‘CK pathway’, ‘RNA modification’, ‘macromolecule methylation’, ‘DNA metabolism’, ‘ATP activity’, and ‘S-adenosylmethionine-dependent methyltransferase activity’ were upregulated . At the transition from D2 to D4, ‘monosaccharide metabolism ’, ‘polysaccharide metabolism ’, ‘lipid catabolism’, ‘amino acid metabolism ’, ‘Sadenosylmethionine biosynthesis’, and ‘L-phenylalanine biosynthesis’ were significantly upregulated . ‘Response to auxin stimulus’ was also significantly upregulated .

Down regulated GO terms were mainly ‘nicotianamine metabolism and biosynthesis’ .At the transition from D4 to D7, ‘iron ion binding’ was significantly upregulated. The only down regulated biological process was the auxinmediated signaling pathway . The over representation of ‘ribosome and cytosolic small ribosomal subunit’ was also enriched in the down regulated GO group .ET is a key flower senescence promoting hormone in ET-sensitive species. In this study, transcriptional dynamics at four distinct developmental stages of corollain petunia were monitored. The ‘S-adenosylmethionine biosynthesis’ GO term was significantly upregulated at the D2 and D4 transition . Expression of ACS and ACO genes was upregulated through D2 to D4 and D4 to D7 transitions. The increase of ET emission was initially detected at the D2 to D4 transition, while the spike of ET emission occurred at the D4 to D7 transition . These data suggest that early onset of corolla senescence may occur in the transition from D2 to D4, and execution of senescence takes place in the transition from D4 to D7.Notably, large alterations in abundances of auxinrelated transcripts occurred throughout the four developmental stages, especially through the transition from D2 to D4. Although, at present, the role of auxin in plant senescence remain poorly defined, and contrasting observations have been obtained from different species. Several studies have reported an involvement of auxin in the process of senescence, especially in petal senescence. For example, in cut carnation flowers, exogenous application of IAA hastened the rise in ET production and flower wilting. 2,4-dichlorophenoxyacetic acid , a synthetic auxin, induced the expression of ACC synthase genes in the styles, ovaries, and petals. It was reported that in the corollas of pollinated petunias, ‘response to auxin stimulus’ and ‘response to ACC’ were significantly enriched at 12 hap. Interaction between auxin and ET occurred at the early stage of pollination. Furthermore, the interaction between ET and auxin was also reported in ET-induced corolla senescence in petunia. Interestingly, during pear ripening, the auxin-associated transcripts are significantly upregulated in the S2 to S3 transition before pear ripening and down regulated in the S3 to S4 transition. In addition, auxin level declined prior to ripening in tomato, grape, and strawberry fruit. Moreover, the largest number of DEGs related to auxin were observed in the abscission process of rose petal. Down regulation of RhIAA16 by VIGS in rose promoted petal abscission. In our transcriptome data, DEGs in the auxin pathway, including auxin-responsive genes , auxin-induced genes , and auxin efflux carrier were all induced at the D2 to D4 transition, where ET production was increased. However, those auxin-related genes were down regulated in the D4 to D7 transition , while expression of ACO and ACS genes was upregulated and ET production reached a peak at 5.5 days . Taken together, we postulate that auxin might play common and vital positive roles in activating ET production and regulating developmental process that lead to subsequent attainment of ripening, senescence, and abscission capacity.Ecolabels are part of a new wave of environmental policy that emphasizes information disclosure as a tool to induce environmentally conscious behavior by both firms and consumers. The goal of ecolabels is to provide easily understood information and thereby elicit increased demand for products perceived as environmentally friendly. An important concern among consumers is that ecolabeled products might entail a trade-off between product quality and environmental impact. In other words, in order to achieve low environmental impact, green products would have to be of lower quality. In this study, we use the case of ecocertification in the wine industry to test the link between environmentally friendly production and product quality.

Most lettuce varieties are considered compact and have a relatively short stature. They are often harvested when they reach a certain size, rather than allowing them to grow to their maximum height. In an NFT system, the focus is usually on growing lettuce for its leaves rather than allowing it to bolt and produce a flowering stalk.

On average, lettuce plants in an NFT system typically reach a height of 20 to 30 centimeters (8 to 12 inches) before they are harvested. At this stage, the outer leaves are usually harvested, and the plant continues to produce new leaves from the center.

It’s worth noting that there are different lettuce varieties available, including compact or mini varieties, as well as larger, more elongated varieties. The specific lettuce variety you choose to grow in your NFT system may affect its height and growth characteristics.

Ultimately, the height at which lettuce grows in an NFT hydroponic system will depend on the specific variety, environmental conditions, and the desired stage of harvest. Regular harvesting and pruning of outer leaves will help maintain the plant’s height and promote continued growth.

Materials needed:

• PVC pipes (4-6 inches in diameter)
• PVC fittings (elbows, tees, couplers)
• PVC glue
• Net pots or cups
• Growing medium (such as perlite, vermiculite, or coconut coir)
• Submersible pump
• Tub or reservoir for nutrient solution
• Air pump and air stones (optional)
• Timer (for pump control)

Step-by-step instructions:

1. Design your system: Determine the size and layout of your hydroponics system based on available space and the number of plants you want to grow. Plan for the main pipe, vertical growing tubes, and nutrient delivery system.
2. Assemble the frame: Use PVC pipes and fittings to create the frame of your system. Construct a sturdy base and vertical supports to hold the growing tubes. Ensure the frame is level and stable.
3. Create the vertical growing tubes: Cut the PVC pipes into sections of suitable length for your plants. These pipes will hold the net pots or cups. Drill holes evenly spaced along the length of each pipe, ensuring they are large enough to accommodate the net pots.
4. Attach the growing tubes: Attach the vertical growing tubes to the frame using PVC fittings. Use elbows, tees, and couplers to create the desired arrangement. Ensure a slight angle for nutrient flow back to the reservoir.
5. Install the nutrient delivery system: Connect the submersible pump to the tub or reservoir. Attach PVC pipes or hoses from the pump to the top end of the vertical growing tubes. This will allow the nutrient solution to flow down through the pipes and reach the plants’ roots.
6. Add net pots and growing medium: Place net pots or cups into the holes drilled in the vertical pipes. Fill them with a suitable growing medium such as perlite, vermiculite, or coconut coir. Insert your plant seedlings or clones into the growing medium, ensuring the roots reach the nutrient solution.
7. Set up the nutrient solution: Mix the appropriate hydroponic nutrient solution according to the manufacturer’s instructions. Fill the reservoir with the solution, making sure the pump is fully submerged.
8. Optional: Incorporate an air pump and air stones into the reservoir to improve oxygenation of the nutrient solution. This can benefit the roots and overall plant health.
9. Test the system: Turn on the pump and check the flow of the nutrient solution through the pipes. Adjust the pump’s flow rate if necessary to ensure proper irrigation and drainage.
10. Maintain and monitor: Regularly check the pH and nutrient levels in the reservoir, making adjustments as needed. Monitor plant growth, and prune or trellis the cucumber vines as they grow.

Remember to research and understand the specific requirements of the plants you intend to grow. Hydroponic systems require proper monitoring, maintenance, and adjustment of environmental factors to ensure successful plant growth and development.

1. Variety Selection: Choose tomato varieties that are well-suited for greenhouse cultivation and have high yield potential. Look for varieties that are disease-resistant, have a compact growth habit, and produce large, flavorful fruits.
2. Nutrient Management: Hydroponic systems require careful monitoring and management of nutrient solutions. Maintain proper nutrient balance by regularly testing and adjusting the solution’s pH, electrical conductivity (EC), and nutrient levels. Follow the recommended nutrient guidelines specific to tomatoes and adjust as necessary throughout the plant’s growth stages.
3. Irrigation: Provide consistent and controlled irrigation to the plants. Dutch buckets typically use a drip irrigation system. Ensure the irrigation system delivers water and nutrients directly to the plant’s root zone while maintaining proper moisture levels and avoiding waterlogging. Monitor and adjust irrigation frequency and duration based on plant needs, environmental conditions, and stage of growth.
4. Pruning and Training: Pruning and training tomato plants in a hydroponic system are essential for maximizing yields and managing plant vigor. Use the “indeterminate” or “vining” tomato varieties suitable for training vertically. Remove suckers (side shoots) and redirect the plant’s energy towards fruit production. Use trellising or string systems to support and train the plants as they grow.
5. Temperature and Humidity Control: Tomatoes thrive in specific temperature and humidity ranges. Maintain optimal greenhouse conditions to ensure healthy growth and fruit development. Monitor and control temperature, ventilation, and humidity levels to prevent stress, diseases, and pests. Aim for temperatures between 65-80°F (18-27°C) during the day and slightly cooler at night.
6. Lighting: Supplemental lighting can be beneficial in extending the growing season and improving yields, especially in regions with limited sunlight. Use high-quality, full-spectrum LED grow lights to provide the necessary light intensity and spectrum for optimal plant growth and fruiting.
7. Pollination: In a greenhouse environment, natural pollination may be limited. Increase fruit set and yield by manually shaking the plants gently or using vibrating devices to facilitate pollination. Alternatively, introduce bumblebees or other pollinators to the greenhouse.
8. Pest and Disease Management: Implement an integrated pest management (IPM) program to monitor and control pests and diseases effectively. Regularly inspect plants for common issues like aphids, whiteflies, spider mites, and diseases such as powdery mildew or tomato leaf spot. Use biological controls, organic insecticides, or fungicides as needed.
9. Harvest and Crop Rotation: Harvest tomatoes at the correct stage of ripeness to ensure optimal flavor and quality. Remove spent plants promptly to prevent the spread of diseases. Follow a crop rotation plan to minimize the risk of soil-borne diseases and maintain soil health.
10. Record-Keeping and Continuous Improvement: Maintain detailed records of your hydroponic system,dutch bucket for tomatoes including nutrient management, pruning techniques, yields, and other key parameters. Regularly analyze the data to identify areas for improvement and optimize your practices accordingly.

Remember that successful tomato production in a hydroponic Dutch bucket system requires careful monitoring, attention to detail, and regular adjustments based on the specific needs of your plants. Continually educate yourself, seek advice from experienced growers, and adapt your practices as you gain more experience in this specialized growing method.

1. Material: Plastic pots are lightweight, durable, and cost-effective. They are available in various sizes, shapes, and colors, offering versatility for different plant species and aesthetic preferences.
2. Drainage Holes: The drainage holes in plastic pots allow water to flow freely, ensuring that excess moisture doesn’t accumulate around the plant roots. These holes prevent the soil from becoming waterlogged and promote healthy root development.
3. Sizes and Shapes: Plastic plant pots with drainage holes come in a range of sizes, from small pots suitable for herbs and small plants to large pots for shrubs and trees. You can find round, square, rectangular, and even specialized shapes to accommodate different planting needs.
4. Saucers and Trays: When using wholesale plant containers with drainage holes, it’s common to pair them with saucers or trays to catch the excess water that drains out. This helps protect your floors or surfaces and allows you to reuse the drained water if desired.
5. Considerations: While plastic pots with drainage holes are popular, it’s important to note that plastic doesn’t provide as much breathability as other materials like clay or terracotta. This can affect moisture retention and airflow to the roots. However, proper watering practices and appropriate soil choice can help mitigate these issues.

When selecting plastic plant pots with drainage holes, consider factors such as the size and type of plant you’re growing, the amount of drainage needed, and the overall aesthetic you desire. These pots are widely available at garden centers, nurseries, and online retailers, offering a convenient and practical option for container gardening.

1. Scale and layout: Commercial NFT systems can cover large areas, utilizing multiple channels or gutters for growing plants. The layout is carefully planned to make the most efficient use of space and allow for easy access, maintenance, and harvesting.
2. NFT channels: The channels used in commercial systems are typically made of durable materials such as PVC or food-grade plastic. They are designed to provide a shallow film of nutrient-rich water that flows over the roots of the plants. The channels may have specific shapes or contours to optimize the flow and prevent pooling or uneven distribution of the nutrient solution.
3. Water and nutrient delivery: A high-capacity water pump is used to circulate the nutrient solution from a central reservoir to the NFT channels. The flow rate is carefully controlled to ensure a thin, uniform film of the solution along the channels. The nutrient solution is typically well-balanced and adjusted based on the specific requirements of the crops being grown.
4. Automation and control systems: Commercial NFT systems often incorporate advanced automation and control systems to monitor and regulate various parameters. These systems can control the nutrient solution flow, adjust pH and EC levels, manage lighting and climate control, and collect data for analysis and optimization.
5. Climate control: Commercial NFT systems often operate in controlled environments, such as greenhouses or indoor facilities. Climate control systems are employed to maintain optimal temperature, humidity, and ventilation levels for plant growth. This includes using heaters, fans, evaporative cooling, and dehumidifiers to create the ideal conditions for the crops.
6. Lighting: Depending on the location and natural light availability, commercial NFT systems may use supplemental lighting to ensure consistent and adequate light for plant growth. High-intensity discharge (HID) lamps or LED grow lights are commonly used to provide the specific light spectrum required by the plants.
7. Monitoring and maintenance: Commercial systems incorporate monitoring and sensor technologies to track important parameters such as pH, electrical conductivity (EC), temperature, and humidity. These systems help growers identify and address any issues promptly. Regular maintenance, including cleaning the channels, checking for clogs, and ensuring proper nutrient solution concentration, is critical for system performance and plant health.
8. Crop selection: Commercial NFT systems are versatile and can be used to grow a wide range of crops, including leafy greens, herbs, strawberries, and certain fruiting crops. The choice of crops depends on market demand, profitability, and the suitability of the system for the specific plants.
9. Workforce and management: Commercial NFT systems require skilled personnel for day-to-day management, including planting, crop maintenance, harvesting, and system maintenance. Additionally, a management team oversees the operation, monitors crop health, and ensures proper nutrient management and system optimization.

Commercial NFT hydroponic systems offer several advantages, such as high-density planting, water efficiency, reduced use of pesticides, and the ability to grow crops year-round in controlled environments. However, setting up and managing a commercial system requires significant investment, expertise, and a thorough understanding of the specific crop requirements and market dynamics.

Results suggest that trees in the HLB world have higher specific micronutrient requirements than what are currently being recommended. In the field, we have several experiments that involve controlled release, ground applied fertilizers; these include the St. Helena project near Dundee, where sweet orange trees on numerous rootstocks are being grown completely with CRF and daily irrigation. The trees will be 7 years old in April, and they are now more than 80% infected with HLB, yet the large majority of the trees across all rootstocks have remained productive. Evolution of the CRF formulas and effects on tree health from St. Helena and other minor field CRF experiments will be discussed. It is clear that a constant supply of nutrients year-round is required to maintain productivity in the HLB world. Additional fine tuning of fertilizer composition, type, and delivery method should lead to improved tree health and productivity. Improved ground nutrition will continue to play a key role in integrated approaches to controlling HLB. Genetic variability for HLB tolerance/resistance is being identified in existing experimental rootstock germplasm planted throughout Florida, with both sweet orange and grapefruit scions. New rootstocks are being identified in these trials that show a reduced infection frequency, and less severe symptoms once infected, as compared to commercial rootstocks. Rootstocks showing promise include complex tetraploids, diploid citranges, and diploid pummelo x mandarin hybrids. Several of these rootstocks have been ‘Fast Track’ released for large-scale commercial evaluation. Current focus is on the identification of rootstocks that can sustain or increase productivity under heavy HLB pressure.

Data on these promising rootstocks will be presented. The fact that there is genetic variability in rootstock germplasm not pre-screened for HLB tolerance/resistance suggests that even greater progress can be made by focused selection, especially from crosses utilizing emerging HLB tolerant/resistant parents. Thus, we have adjusted our rootstock breeding/greenhouse screening program to focus on HLB by developing the ‘Gauntlet’ screening program described below. Following a preliminary calcareous soil/Phytophthora screen, selected individual hybrid rootstock candidates are transferred to citripots in commercial potting soil. Tops of these trees are propagated by rooted cuttings to produce seed trees on their own roots. The remaining individual liners are grafted with HLB-infected budsticks of Valencia sweet orange. The remaining rootstock top is then removed, forcing flush from the HLB-infected budstick. Trees are monitored for HLB symptoms, and healthy appearing trees are entered into a ‘hot psyllid’ house until psyllid feeding damage is observed on their leaves , followed by field planting at a challenging field site .The oldest ‘Gauntlet’ trees have now been in the field for approximately 2 years, and 20 promising new rootstocks have been identified so far. Our goal is to develop rootstocks that will facilitate sustainable and profitable citriculture in an HLB-endemic Florida, and possibly eliminate the need for psyllid control. Huanglongbing is one of the most devastating citrus diseases in the world. In Florida, it is associated with a bacterium Candidatus Liberibacter asiaticus and transmitted by a psyllid, Diaphorina citri Kuwayama.

We tested D. citri collected in many different venues over a period of 6 years for Las by molecular methods. Results surprised us. first, positive D. citri can be found long before symptoms develop on the plants at the site. Second, positive psyllids can ride on unprocessed fruit in trailers, even when there is no foliage. Third, about 10% of psyllid samples collected from plants for sale in Florida tested positive for Las. Finally, our data, and a related mathematical model, predict a form of transmission of Las that vastly increases the potential for spread of HLB. The mechanism now is known. The increase of infected vectors follows the growth of the insect population, independent of the incubation period in the plant. The implications of this new mechanism completely change our understanding of the epidemiology of HLB. It is possible to have positive D. citri throughout a grove before ever seeing a symptomatic plant. This mechanism has profound implications for disease spread, epidemiological research, early detection, long range dispersal, and grove management. Through utilizing the nutrient-rich phloem sap, sap feeding insects such as psyllids, leaf hoppers, and aphids can transmit many phloem-restricted pathogens. On the other hand, multiplication of phloem-limited, uncultivated bacteria such as Candidatus Liberibacter asiaticus inside the phloem of citrus indicates that the sap contains all the essential nutrients needed for the pathogen growth. Genome sequencing studies revealed that CLas can metabolize many sugars and amino acids found in the phloem sap. In addition, CLas can act as energy parasites and scavenge ATP from its host through the use of an ATP/ADP translocase. Furthermore, reduction in some minerals such as Zn and P in CLas-infected trees indicated that these minerals are required for the growth of CLas. The presence of gene in CLas genome also indicated that CLas can import Zn from its host. The phloem sap composition of many plants has been studied; however,drainage pot available data about citrus phloem sap is limited. In this study, we investigated the phloem sap composition of sweet orange. The phloem sap collected by EDTA or centrifugation method was derivatized with three different reagents and analyzed with GC-MS revealing 20 amino acids, 8 sugars, and 8 organic acids. Analysis of citrus phloem sap by inductive coupled plasma showed that it was rich in potassium, calcium, phosphorus, magnesium, and sulfur. Trace amounts of iron, copper, zinc, and boron were also detected. The ATP concentration in citrus phloem was 24.0 ± 4.0 ppm. Analysis of citrus phloem sap high performance liquid chromatography showed that citrus phloem sap was rich in nucleotides. Studies on seed transmission of ‘Candidatus Liberibacter asiaticus’ , the bacterium associated with Huanglongbing , described seedlings from infected trees which showed an abnormal growth phenotype but were free of CLas. We germinated populations of seeds of ‘Hamlin’, ‘Ridge Pineapple’, and ‘Valencia’ sweet orange from infected trees and observed chlorotic and stunted seedlings which failed to grow substantially. Tests detected no CLas DNA in these seedlings. These aberrant phenotypes were similar to symptoms expressed by infected trees, and persisted for 6 months or longer, at which point seedlings died or began to grow normally. Normal growth also was induced by grafting stunted seedling apices to healthy citrus seedlings. The absence of infection and their HLB-symptomatic phenotype suggest that extracellular factors produced by CLas in the mother tree affected the normal development of these seedlings, possibly through the alteration of normal gene expression. The similarity of these seedlings and symptomatic foliage suggest these factors are involved in disease development in infected trees. We propose these seedlings are a model system for understanding the molecular basis of symptom development in HLB-affected citrus trees.As Huanglongbing has continued to spread across the state, there is a growing body of evidence to support that in some instances, there are some off-flavors that can be detected in juice coming from fruit of HLB-infected trees compared to juice coming from the fruit of healthy trees.

The differences tend to be subtle but detectable in sensory evaluations by trained panelists, and in some instances by untrained panelists. Among the descriptors that have been used to describe HLB juice are bitter, sour, astringent, metallic, salty, umami, less orange aroma, less orange mouth feel, etc. Similarly, multiple studies have also identified several juice quality parameters, metabolites, or compounds that change with HLB infection; but, in all cases it has been changes in levels and not the presence or absence of a specific chemical. For instance, juice from HLB-infected fruit results in lower Brix, higher acid, and higher levels of limonin and nomilin. However, there is no one “smoking gun” parameter that can be measured that would indicate the potential for off flavors. In the present study, 14 different parameters were evaluated that encompassed sensory descriptors , basic juice chemistry , secondary metabolites , and harvest associated parameters . Samples were collected at random from fruit loads delivered to a commercial processing plant and were extracted using commercial equipment by the “USDA/State” lab that exists at commercial processing facilities. Sensory data were collected through the use of an electronic tongue. Basic juice chemistry data were collected using traditional wet chemistry techniques. Secondary metabolites were measured by HPLC and harvest date and fruit size were collected at the processing facility. Samples were collected from both Hamlin and Valencia varieties. Principal component analysis was used to reduce the number of variables considered for each of the varieties and the results were compared to a novel qPCR method to cross check the conclusions. Initial results indicated that a subset of parameters that include some sensory, basic juice chemistry, secondary metabolites, and harvest data can be used to identify fruit loads that have the potential to produce off flavors in Hamlin and to a lesser extent in Valencia varieties.Since its discovery in Florida in late 2005, citrus Huanglongbing , widely recognized as the most serious disease of citrus, has caused havoc in the Florida industry. Production costs have increased and production is down. Despite the apparent advances that growers and researchers have made in mitigating some of the symptoms of the disease, the statewide production continues to go down. All of the variables that go into the state forecast, tree number, fruit size, fruit per tree, and percent fruit drop are all trending in the wrong direction. This has led some growers to hold back on replanting in recent years which has further exacerbated the problem. The big question is how much lower will production go? Based on what we know, or in some cases based on our best guess, a model was created to estimate where we are in the HLB decline curve in the Florida industry. The model assumes that the industry focus will continue on the track of symptom mitigation instead of inoculum removal and management. Based on some conservative assumptions used in the model, the significant losses that have occurred the last several years should have been predicted. Going forward with the same assumptions, it appears that the production decline is near its predicted lowest significant drop and future losses will track the rate of replanting. That is to say, if tree attrition is greater than the rate of replanting, production will continue to decline. If the replanting rate is equal to or greater than the attrition rate, then the production will hold or increase slightly over time. Citrus is one of the most economically important and extensively grown fruit tree crops worldwide. Citrus production in most citrus producing countries, e.g., US, Brazil, and China, is facing an unprecedented challenge caused by Huanglongbing . Currently, no effective HLB management is available. Development of HLB resistant or tolerant citrus will provide a long-term, effective, and sustainable solution to HLB. Traditional plant breeding is unlikely to lead to HLB resistant or tolerant plants due to the lack of resistant varieties. Targeted genome engineering is expected to contribute significantly to future varietal improvement. Genome editing technologies using zinc finger nucleases , transcription activator-like effector nucleases , and clustered regularly interspaced short palindromic repeat /Cas9/single guide RNA have already been successfully used to genetically modify plants. Here, we reported our progress in modifying citrus genome suing Cas9/sgRNA technology.

Studies of bacterial pathogens in perennial crops have evaluated the utility of pruning as a disease management tool, with mixed results . The removal of infected plant tissues is analogous to measures used for management of trunk diseases, often referred to as “remedial surgery,” as an alternative to replacing infected plants . In this study, we investigated whether severe pruning of Xylella fastidiosa-infected grapevines in commercial vineyards could clear vines of existing infections. Pierce’s disease is a lethal vector-borne disease of grapevines caused by the bacterium X. fastidiosa . After susceptible plants are inoculated by X. fastidiosa, pathogen populations multiply and move through the xylem network, leading to symptoms of reduced water flow , including leaf scorch, cluster desiccation, vine dieback, and eventually death. There is no cure for grapevines infected with this bacterium; current strategies for management of PD in California vineyards involve limiting pathogen spread to uninfected vines by controlling vector populations, disrupting transmission opportunities, and eliminating pathogen sources in the surrounding landscape . PD is notable for the numerous sources of variability in infection levels and symptom severity in plants. X. fastidiosa infection levels vary among plant species , grapevine cultivars , seasons , and as a function of temperature . Like other bacterial plant pathogens , X. fastidiosa is often irregularly distributed within individual hosts.

For example, X. fastidiosa infection levels in grapevines may vary by more than 10-fold between grapevine petioles and stems ; in other hosts, infection levels may vary by more than 100-fold between basal and apical sections of shoots . This within-host heterogeneity may be epidemiologically significant if it affects pathogenacquisition efficiency . Moreover, if such variation is associated with protracted localized infection near inoculation points, such heterogeneity may facilitate other disease management tactics. In addition to grapevines, other plant species that are susceptible to X. fastidiosa infection include citrus in South America . Management of the resulting disease in C. sinensis relies on clean nursery stock, vector control, and pruning infected plant tissue from established trees or roguing young plants . The concept of pruning of infected plant material is based on the fact that, in established trees , tissue with early symptoms of infection can be pruned ~1 m proximal to the most symptomatic basal leaf, effectively eliminating infections, as the remaining tissue is free of X. fastidiosa. However, pruning is not adequate for young trees or for removing bacterial infections if any symptoms are present in fruit . X. fastidiosa multiplies and spreads through the xylem vessels, reaching the roots of perennial hosts such as citrus , peach , alfalfa , and blueberry . Nonetheless, under field conditions, chronic infection of grapevines is temperature and season dependent. In regions with freezing winter temperatures, infected plants can recover in winter, curing previously infected and symptomatic grapevines .

Infections that occur during spring lead to chronic disease ; however, infections that occur during late summer and fall may cause disease symptoms in the current year, but a high proportion of vines lack symptoms of X. fastidiosa infection in the following year .Nonetheless, models that incorporate low temperatures have substantial explanatory power in predicting rates of winter curing of X. fastidiosa infections in grapevine . Infections that occur early in the season may have a longer period during which X. fastidiosa can colonize and reach high infection levels, which may increase the likelihood of the disease surviving over the winter. Following this rationale, if most late-season infections remain in the distal ends of shoots and have lower infection levels, removing the symptomatic portion of the vine might eliminate X. fastidiosa. In other words, the efficacy of pruning infected grapevine tissue could depend on both the time of year in which the plant was infected and on winter temperature. A potential benefit of severe pruning versus replanting is that pruning leaves a mature root stock in place, which is likely to support more vigorous regrowth compared to the developing root stock of a young transplant . Recent attempts to increase vine productivity by planting vines with more well-developed root systems are based on this presumption. However, even if severe pruning can clear vines of infection,drainage pot it removes a substantial portion of the above ground biomass of the vine. Thus, a method for encouraging rapid regrowth of the scion after aggressive pruning is needed. We studied the efficacy of pruning infected vines immediately above the root stock graft union—the most aggressive pruning method—for clearing grapevines of infection by X. fastidiosa. We reasoned that if such severe pruning was ineffective at clearing vines of infection, less severe pruning would not be warranted; if severe pruning showed promise, less severe pruning could then be tested. We use the term “severe pruning” to refer to a special case of strategic pruning for disease management, analogous to the use of “remedial surgery” for trunk diseases . To test the efficacy of clearing vines of X. fastidiosa infection, we followed the disease status of severely pruned versus conventionally pruned vines over multiple years, characterized the reliability of using visual symptoms of PD to diagnose infection, and compared two methods of restoring growth of severely pruned vines.Pruning trials were established in Napa Valley, CA in commercial vineyards where symptoms of PD were evident in autumn of 1998. The vineyards used for these trials varied in vine age, cultivar, and initial disease prevalence . All study vines were cordon-trained and spur-pruned. We mapped the portions of the six vineyards selected for study according to evaluation of vines for disease symptoms. The overall severity of PD symptoms for each vine was recorded as follows: 0 = no symptoms, apparently healthy; 1 = marginal leaf scorch on up to four scattered leaves total; 2 = foliar symptoms on one shoot or on fewer than half of the leaves on two shoots on one cordon, no extensive shoot die back, and minimal shriveling of fruit clusters; and 3 = foliar symptoms on two or more shoots occurring in the canopy on both cordons; dead spurs possibly evident along with shriveled clusters.

To test the reliability of the visual diagnosis of PD, petiole samples were collected from the six vineyard plots when symptom severity was evaluated for vines in each symptom category; these samples were assayed using polymerase chain reaction . Petioles were collected from symptomatic leaves on 25, 56, and 30 vines in categories 1, 2, and 3, respectively. Next, severe pruning was performed between October 1998 and February 1999 in the six vineyard plots by removing trunks of symptomatic vines ~10 cm above the graft union. Cuts were made with saws or loppers, depending upon the trunk diameter. During a vineyard survey, severe pruning was conducted on 50% of vines in each symptom category; the other 50% of vines served as conventionally pruned controls. Sample sizes for control and severely pruned vines in each disease category ranged between six and 62 vines depending on the plot, with at least 38 total vines per plot in each control or pruned treatment. In spring 1999, multiple shoots emerged from the remaining section of scion wood above the graft union on severely pruned vines. When one or more shoots were ~15 to 25 cm long, a single shoot was selected and tied to the stake to retrain a new trunk and cordons, and all other shoots were removed at this time. We evaluated the potential of severe pruning to clear vines of infection, by reinspecting both control and severely pruned vines in all six plots for the presence or absence of PD symptoms in autumn 1999 and 2000. In all plots, category 3 vines were inspected in a third year ; in plot 6, vines were inspected an additional two years . Finally, in plot 6 we investigated chip-bud grafting as an alternate means of ensuring the development of a strong replacement shoot for retraining. To do this, 78 category 3 vines were selected for severe pruning, 39 of which were subsequently chip-bud grafted in May 1999. An experienced field grafter chip budded a dormant bud of Vitis vinifera cv. Merlot onto the rootstock below the original graft union, and the trunk and graft union were removed. The single shoot that emerged from this bud was trained up the stake and used to establish the new vine. The other 39 vines were severely pruned above the graft union and retrained in the same manner as vines in plots 1 to 5. Development of vines in plot 6, with and without chip-bud grafting, was evaluated in August 1999 using the following rating scale: 1) “no growth”: bud failed to grow, no new shoot growth; 2) “weak”: multiple weak shoots emerging with no strong leader; 3) “developing”: selected shoot extending up the stake, not yet topped; and 4) “strong”: new trunk established, topped, and laterals developing. All analyses were conducted using R version 3.4.1 . We used a generalized linear model with binomial error to compare the relative frequency of X. fastidiosa-positive samples from vines in the different initial disease severity categories . Next, we analyzed the effectiveness of chip budding versus training of existing shoots as a means for restoring vines after severe pruning. This analysis used multinomial logistic regression that compared the frequency of four vine growth outcomes the following season: strong, developing, weak, or no growth. This main test was followed by pairwise Fisher exact tests of the frequency of each of the individual outcomes between chip budded-trained and trained vines . We analyzed the effect of severe pruning on subsequent development of PD symptoms using two complementary analyses. first, we compared symptom return between severely pruned and control vines in the three symptom severity categories for two years after pruning. To appropriately account for repeated measurements made over time, our analysis consisted of a linear mixed-effects model with binomial error, a random effect of block, and fixed effects of treatment , year , and symptom severity category . Next, we analyzed the rate at which PD reappeared in only severely pruned vines from category 3 in subsequent years using a survival analysis. Specifically, we used a Cox proportional hazards model with a fixed effect of plot .Accurate and time- or cost-efficient methods of diagnosing infected plants are important elements of a disease management program, both with respect to roguing to reduce pathogen spread , and the efficacy of pruning to clear plants of infection . Accurate diagnosis of PD in grapevines is complicated by quantitative and qualitative differences in symptoms among cultivars and other aspects of plant condition . Our results suggest that a well-trained observer can accurately diagnose PD based on visual symptoms, particularly for advanced cases of the disease. The small number of false positives in disease category 1 and 2 vines may have been due to misdiagnosis of other biotic or abiotic factors . Alternatively, false positives might indicate bacterial populations that are near the detection limit; conventional PCR has at least as low a detection threshold as other methods that rely on the presence of live bacterial cells .

PKs may play a key regulatory role in drought stress adaptation in M. flabellifolia, as corroborated by comparison of our data with Arabidopsis and chrysanthemum drought stress expression profile data.In Arabidopsis, only 121 PKs were found to be responsive to osmotic stress.Xu et al. identified only 229 differentially expressed PKs during drought stress in chrysanthemum, a species with a large and complex polyploid genome and high heterozygosity.Protein family distribution analyses showed that three receptorlike kinase type families – leucine-rich repeat kinases, domain of unknown function DUF, and receptor like cytoplasmic kinases – were the largest groups of DTGs in both the dehydration and rehydration treatments . RLKs constitute the largest PK family in plants and have been implicated in the regulation of meristem proliferation, organ specification, reproduction, and hormone signal transduction.Drought transcript profiling in Arabidopsis has revealed that the transcript abundance of many RLK genes peaks 1 h after the start of drought treatment, indicating that RLKs may also be part of a rapid drought response.Several functional studies have also confirmed roles of RLKs in drought tolerance.In Arabidopsis, an LRR kinase, receptor protein kinase 1 , is induced by ABA. Repression of RPK1 decreases sensitivity to ABA, suggesting that RPK1 is involved in ABA perception.A DUF type kinase, CRK, and a receptor-like cytoplasmic type kinase, ARCK1, form a complex that negatively controls ABA and osmotic stress signal transduction.

In addition,DTGs encoding mitogen-activated protein kinases were identified during dehydration stress in M. flabellifolia . MAPK cascades function in transducing environmental and developmental cues to intracellular responses.In Arabidopsis, several MAPKs are involved in abiotic stress signaling pathways including MEKK1,MPK1,MPK3,MPK4,MPK6,MAP9,and MAP12.The transcript abundance of homologs of all these MAPKs, apart from MPK6 and MAP12, was altered in response to dehydration in M. flabellifolia . To identify early responsive regulatory genes, the expression pattern of differentially transcribed TFs and PKs was analyzed by hierarchical clustering . The transcript abundance of 53 TFs and 91 PKs peaked early in dehydration , indicating that broad regulatory networks are quickly employed to coordinate global transcriptional reprogramming during desiccation of M. flabellifolia. Among the up-regulated TFs during early dehydration, the strongest induction was observed for genes encoding putative DREB family members . The DREB TFs are well-known regulators of ABA-independent drought stress signaling pathways in other plant species.In addition, a total of nine genes encoding WRKY family members were included among the up-regulated TFs in early dehydration, making the WRKY family the biggest group of early dehydration up-regulated TFs . Furthermore, among the up-regulated PKs, the LRK10 L kinase family is one of the largest groups in during early dehydration , suggesting that LRK10 L kinases may be important upstream regulators of rapid responses to water deficit. For example, the transcript abundance of a DTG encoding an LRK10 L kinase increased nearly 120-fold in early dehydration .

LRK10 L genes have previously been reported to be associated with resistance to pathogen infection.Functional characterization of these LRK10 L kinases will be an important step toward elucidating their roles in desiccation tolerance.Various genetically encodable reporters have been developed to monitor gene expression, protein subcellular localization, protein stability, hormonal signaling, and impacts of environmental signals. The green fluorescent protein and its derivatives such as RFP, mCherry, and YFP have many applications as reporters for gene expression or as fusion proteins. Although GFP is easy to use, it needs light sources to visualize the fluorescence signals. The β-glucuronidase reporter has been widely used in plants for monitoring gene expression patterns and as a reporter for hormonal signaling. For example, DR5-GUS transgenic lines are commonly used to monitor auxin distribution and auxin signaling. Luciferase is another broadly used reporter in both animals and plants. Both GUS and luciferase require the addition of expensive substrates X-Gluc and luciferin, respectively. Whereas the traditional reporters have been very useful, they have limitations. Fluorescent proteins are often monitored under a microscope, rendering it less useful in analyzing plants in natural growing fields or analyzing large samples such as a tree. GUS staining is invasive and often requires sacrifice of the plants. Luciferase can be used non-invasively, but it requires a special camera and spraying the expensive substrate. It is also not very practical to use them in fields. GUS and luciferase may not be optimal for sterile conditions such as tissue culture because addition of substrates increases the chance for contamination of microbes. Therefore, there is a need to develop new reporter systems that can be widely used to monitor cellular activities noninvasively, continuously, and cost effectively. For the past few years,strawberry pots gene editing has been widely used in basic research and crop improvement. A visible marker for transgenes will greatly accelerate the isolation of edited plants that no longer harbor the gene editing machinery. Plants produce many colorful compounds that potentially can serve as reporters. For example, anthocyanins display bright red-blue colors and anthocyanin-producing rice plants have been used to generate interesting patternsin rice field. However, synthesis of anthocyanins requires multiple enzymes and varies greatly among different plants. It is difficult to use anthocyanin biosynthesis pathways as a universal visible reporter. Betalains are a class of plant natural products derived from the aminoacid tyrosine. The bright red color seen in beets, dragon fruit, Swiss chard, and other plants is resulted from accumulation of betalains. Biosynthesis of betalains has been well studied and only needs three enzymatic reactions to convert tyrosine into betalain. Tyrosine is first hydroxylated on the benzene ring, resulting in L-3,4-dihydroxyphenylalanine . The reaction is catalyzed by the P450 oxygenase CYP76AD1 . L-DOPA can be further oxidized into cyclo-DOPA by CYP76AD1 . Alternatively, LDOPA is catalyzed by L-DOPA 4,5-dioxygenase into betalamic acid, which is subsequently condensed with cyclo-DOPA into betanidin. The condensation reaction does not require an enzyme . Finally, a sugar moiety is added to betanidin by a glucosyltransferase to generate the colorful betalain . Betalain has a very bright red color, which potentially can serve as a reporter to track gene expression or to visualize transgenic events. Because every cell contains the amino-acid tyrosine, exogenous application of tyrosine to tissues may not be required. We hypothesized that betalain would be a more convenient reporter than the aforementioned reporters. It is visible to naked eyes without any needs for special equipment. It does not require processing samples and it allows continuously monitoring events throughout the life cycle of an organism. Moreover, it is applicable to large plants grown under normal field conditions. Herein, we synthesize an artificial open reading frame named RUBY that when expressed can produce all of the enzymes required for betalain biosynthesis. We show that RUBY is a very effective marker for noninvasively selecting transformation events in both rice and Arabidopsis. Moreover, we show that RUBY can be used to visualize gene expression without any chemical treatments or special equipment, providing useful tools for visualizing gene expression in large plants under natural field growth conditions.Heterologous expression of CYP76AD1, DODA in tobacco, and other plants demonstrated that the betalain biosynthetic pathway can be re-constituted in plant cells. In order to use betalain as a visual reporter, we need to effectively co-express the entire pathway using a single promoter. We organized CYP76AD1, DODA, and Glucosyltransferase into a single open reading frame . The stop codons of CYP76AD1 and DODA were removed. The three genes were linked by sequences that encode 2A peptides. Upon transcription, the single transcript, which includes the coding regions of the three enzymes, produced the three separate enzymes through either 2A-mediated self-cleavage or ribosomal “skipping”.

The 2A system enables the expression of multiple proteins under the control of a single promoter. We name the 2A-linked unit of CYP76AD1, DODA, and Glucosyltransferase RUBY . RUBY can be expressed when a promoter is placed in front of it. The expression pattern and level of a particular gene may be inferred from the red color of betalain if the gene’s promoter is used to drive RUBY expression.We first placed RUBY under the control of Cauliflower Mosaic Virus 35S promoter, which is a widely used constitutively strong promoter. To test whether RUBY can produce functional enzymes for betalain synthesis, we infiltrated tobacco leaves with Agrobacteria that contain RUBY-expressing plasmid.Transient expression of RUBY led to the production of betalain in tobacco leaves, suggesting that the synthetic open reading frame RUBY can produce the functional enzymes for the synthesis of betalain. Moreover, we observed that betalain was not transported from the spots of Agrobacterium-infiltration spots to other leaves of the plant .We transformed the 35S:RUBY construct into Arabidopsis using Agrobacterium-mediated floral dipping. Two days after floral dipping, we noticed that the transformed plants displayed patches of red color , indicating that the RUBY cassette was functionally expressed and that RUBY may be used to monitor transient Arabidopsis transformation. Once the seeds from the Agrobacterium-dipped plants were harvested, transgenic seeds could be easily differentiated from non-transgenic seeds . The transformed seeds had a dark red color , demonstrating that RUBY can be used as a visual selection marker for transgenic events in Arabidopsis. We previously used mCherry as a very effective marker to select transgenic events , which requires a dissecting microscope with fluoresence capability. RUBY is a better option because it does not require special equipment. The 35S:RUBY plants produced sufficient amount of betalain to become visually evident . Consistent with previous reports that CaMV 35S promoter is constitutively active, we observed red color in all tissues throughout the plant life cycle . We also expressed RUBY reporter under the control of the Maize UBIQUITIN promoter, which has been widely used to over express genes in monocots. Similar to 35S:RUBY plants, UBQ:RUBY plants were also visibly red in leaves, stem, and flowers . These results clearly demonstrated that RUBY could be expressed in Arabidopsis and that our RUBY reporter was able to functionally re-constitute the betalain biosynthetic pathway. We expressed RUBY using the seed specific At2S3 promoter, which we previously used to drive mCherry expression in Arabidopsis to facilitate the selection of transgenes. As shown in Fig. 2c, the transgenic plants were indistinguishable from wild type plants. When we checked the seeds in a silique from an At2S3:RUBY T1 plant, RUBY-expressing seeds displayed strong red color, whereas the non-transgenic seeds were green . RUBY can be conveniently used to select single T-DNA insertion events by analyzing the ratio of red seeds to green seeds, which should be ~3:1 for single insertions. The At2S3:RUBY results demonstrate that RUBY could be an effective marker for Arabidopsis transformation. Furthermore, betalain was not widely transported from the sites of synthesis to other tissues as we did not see any red color in leaves . We also expressed RUBY under the control of the Arabidopsis YUC4 promoter . YUC4, which encodes a key enzyme in auxin biosynthesis, was shown to express in small regions of embryos, leaves, and flowers. GUS signals were observed in leaf tips and apical region of a gynoecium in YUC4 promoter:GUS transgenic plants. We observed similar patterns of betalain production in YUC4:RUBY lines .Unlike Arabidopsis, rice and many other plants are transformed through tissue culture and the formation of calli, which are often mosaic.

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.