Progress 11/01/09 to 11/01/12
Outputs
OUTPUTS: A majority of fruit tree crops produce fruits in ON and OFF years, which is referred as alternate bearing (AB). Avocado (Persea americana) production is a multi-million dollar industry in the United States and California produces 90% of the avocados in the U.S. Due to the AB habit of avocado, fruit production declines in the OFF years, which results in millions of dollars of lost revenue and creates a monetary strain on the growers in California. Current hypotheses indicate that shoots produce an AB-signal to reduce the flowering potential of the shoot, in order to maximize nutrient flow to fruits. This AES proposal is aimed at obtaining a global proteomic map from vascular sap (phloem and xylem) in ON (trees with fruit) and OFF (trees without fruit) trees in order to identify a mechanism by which fruit load alters the growth and flowering potential of the shoot apical meristem. Next, we will determine if fruit load has a similar effect in a rapid cycling plant such as Arabidopsis. The long-term goal of our research is develop breeding programs to reduce the effect of fruit load on shoot growth and flowering so that breeders and growers effectively mitigate AB in fruit trees crops to improve efficiency and enhance yields in the OFF years of plant development. Outputs 1: Results from our proteomics study in the first and second year, suggested that fruit load alters auxin signaling in the shoot. Auxin gradients and signaling play a fundamental role in shoot growth and development. Therefore, we performed a fate map analysis in ON and OFF shoots of avocado to determine the effect that fruit has on the growth and flowering potential of the shoot. Output 2 and 3: In outputs 2 and 3 we determined if fruit load alters auxin signaling and response in the rapid cycling plant Arabidopsis. Moreover, we examined how fruit load alters the function and fate of the shoot meristem. Results from Output 1 were presented at the Avocado International Brainstorming Meeting, in New Zealand, August 30th, 2011. PARTICIPANTS: This project is collaborative project between my laboratory, Dr. Mary Lu Arpaia, CE Specialist Horticulturist at UCR and Reuben Hofshi, Advisor at the ACW Avocado Ranch in Fallbrook, CA. A Junior Specialist working for Dr. Arpaia, Rodrigo Iturrieta, and graduate students, Shruti Lal and Shang Wu, were valuable contributors to this project. Mr. Iturrieta received his Masters Degree in Avocado Tree Physiology from the Universidad Catholica Valparasio in Chile. To complement Dr. Iturrieta's background in plant physiology, Mr. Iturrieta has received training in developmental biology, biochemistry and proteomic analysis, while working on this project. Mr. Iturrieta is now a graduate student in the Department of Botany and Plant Sciences at UCR. Shurti Lal and Shang Wu received training in proteomics, gene expression analyses and crop tree development. Reuben Hofshi provided us with avocado trees at the ACW avocado ranch to conduct the experiment. Lastly, Jenna Browning-Cammins, a CEPCEB-REU student, participated in the fate mapping analysis this summer. TARGET AUDIENCES: Our target audience includes growers, farm advisors and mangers as well as plant developmental biologists. Other target audiences include the California Avocado Commission and as well as federal funding agencies. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
In the spring of 2011, we performed a fate map analysis of ON and OFF shoots. Results from our analysis show that the apical buds of ON shoots enter a dormant state. First, the fruit completely inhibited shoot growth (spring and summer flush) if the fruit set before the apical bud was released. Second, if the apical bud was released prior to fruit set, 35% fewer nodes were expanded in ON spring shoots compared to OFF spring shoots. Third, fruit load completely inhibited growth of the summer flush in 42% of the shoots bearing fruit. Our fate map analysis showed that the axillary buds of the summer flush had a significantly higher propensity for undergoing floral induction than the axillary buds initiated by the spring flush. Taken together, our results show that fruit load reduces the growth potential of the spring and summer flush. Moreover, the reduction in the shoots ability to initiate a summer flush likely attributes to the negative effect that fruit load has on floral induction. Therefore, we speculate that the primary effect of fruit load on shoot development is an overall inhibition of shoot growth. Experimental studies show that the shoot meristem enters a dormant state in response to fruit load in annuals and perennials. Results from our proteomic study suggested that fruit load alters auxin signaling in avocado shoots. Therefore, we examined the effect of fruit load on auxin signaling in Arabidopsis using DR5:GUS, which measures auxin response. Our results show that DR5:GUS activity is low in actively growing shoots. However, upon fruit load-meristem cessation, strands of DR5:GUS can be detected in the stem and pedicels approximately 10 mm below the shoot apex. Histological studies show that upon fruit load-meristem cessation, files of lignified cells are produced at the base of the shoot meristem. An increase in the number of lignified cells at the base of the shoot meristem was also observed in ON shoots of avocado. Using mRNA in situ hybridization, we determined the effect of fruit load-meristem cessation on meristem function and development. Stem cell homeostasis is regulated in part by CLAVATA3 (CLV3) and WUSCHEL (WUS). During shoot growth, auxin regulates organogenesis in part through MONOPTEROS (MP), an Auxin Response Factor. Lastly, SHOOT MERISTEMLESS (STM) encodes a homeodomain transcription factor that promotes meristem cell fate. In actively growing inflorescence meristems, CLV3 and WUS are expressed in the central zone and rib meristem, respectively, while MP is expressed in the peripheral region of the shoot meristem. Upon fruit load-meristem cessation, transcripts for CLV3, WUS and MP could not be detected suggesting that fruit load somehow negatively regulates the expression of these genes. In contrast to these expression patterns for CLV3, WUS and MP, STM transcripts were detected in active and dormant meristems suggesting that fruit load does not alter the fate of the cells in the meristem.
Publications
- No publications reported this period
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Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: A majority of agricultural tree crops produce fruits and seeds in ON and OFF years, which is referred as alternate bearing (AB). Avocado (Persea americana) production is a multi-million dollar industry in the United States and California produces 90% of the avocados in the U.S. Due to the AB habit of avocado, fruit production declines in the OFF years, which results in millions of dollars of lost revenue and creates a monetary strain on the growers in California. Current hypotheses indicate that shoots produce an AB-signal to reduce the flowering potential of the shoot, in order to maximize nutrient flow to fruits. This AES proposal is aimed at obtaining a global proteomic map from vascular sap (phloem and xylem) in ON (trees with fruit) and OFF (trees without fruit) trees in order to identify mobile proteins that function to inhibit flowering. We refer to these types of proteins as AB-proteins. Once identified, AB genes will be cloned from avocado and transformed into rapid cycling annual plants to determine if ectopic AB gene expression represses flowering. Once AB proteins are characterized, we will analyze these proteins as well as FLOWERING LOCUS T (FT), a universal flower promoting protein, to determine the relationship and the role of these opposite functioning proteins in alternate bearing in avocado as well as other fruit tree crops. The long-term goal of our research is develop breeding programs to reduce the levels and/or activities of AB proteins so that breeders and growers can control alternate bearing in fruit trees crop to improve efficiency and enhance yields in the OFF years of plant development. Outputs 1: In the second year of this proposal, we determined the proteomic profiles for phloem sap proteins in ON and OFF trees at the shoot level. This is an important modification, as not all shoots in a tree bear fruit. In addition, in the Hass cultivar, the AB-signal appears to function at the shoot level and not the tree level. Therefore, it is imperative that sampling be done from the appropriate shoots in ON trees. For this output, phloem sap was collected from leaves of ON and OFF shoots before (June) and during the time of floral induction (August). Phloem sap was also collected from developing fruit using a pressure bomb. Comparative proteomics was used to determine the proteins that are specifically enriched in the vascular sap of ON shoots (shoots bearing fruits). In addition, proteomics was aimed at understanding the biochemical similarities as well as differences between the vascular sap in ON and OFF trees. Output 2 and 3: Outputs 2 and 3 will determine the function of AB proteins using a rapid cycling plant and correlate alternate bearing with the presence or absence of AB protein and gene expression. Based on the results for Objective 1, we have modified our experimental design in order to complete this part of the proposal. Results from Output 1 have been presented to Dr. Mary Lu Arpaia and Reuben Hofshi, Advisor for the ACW avocado ranch. On June 6th, 2010, I presented my initial findings to the California Avocado Commission on Avocado Genetics and Breeding at UCR. PARTICIPANTS: This project is collaborative project between my laboratory, Dr. Mary Lu Arpaia, CE Specialist Horticulturist at UCR and Reuben Hofshi, Advisor at the ACW Avocado Ranch in Fallbrook, CA. A Junior Specialist working for Dr. Arpaia, Rodrigo Iturrieta, and a graduate student, Shruti Lal, were valuable contributors to this project. Mr. Iturrieta received his Masters Degree in Avocado Tree Physiology from the Universidad Catholica Valparasio in Chile. To complement Dr. Iturrieta's background in plant physiology, Mr. Iturrieta has received training in developmental biology, biochemistry and proteomic analysis, while working on this project. Mr. Iturrieta has applied to graduate school, and has been tentatively accepted into the graduate program in the Department of Botany and Plant Sciences at UCR. Shurti Lal, a 5th graduate student in my laboratory, received training in proteomics and crop tree development. Reuben Hofshi provided us with avocado trees at the ACW avocado ranch to conduct the experiment. Mr. Mauricio Tapa, Farm Manager for ACW maintained the trees and he has been a valuable consultant on determining floral bud set in the trees. Lastly, an undergraduate in my laboratory, Dr. Nolan Ung, helped collect samples. Nolan Ung will be participating in the future on modeling auxin gradients in shoots bearing fruits. TARGET AUDIENCES: Our target audience includes growers, farm advisors and mangers as well as plant developmental biologists. Other target audiences include the California Avocado Commission and as well as federal funding agencies. PROJECT MODIFICATIONS: Auxin plays an essential role in plant development. In the shoot, auxin regulates numerous developmental programs including apical dominance. During shoot development, the basipetal transport of auxin from the main shoot apex inhibits the outgrowth of axillary buds, a process known as apical dominance. Physiological and mathematical modeling studies indicate that the establishment of the polar auxin transport system (PATS) from the source (main shoot apex) to the sink (root) dampens the transport of auxin out of the lateral buds. The seeds of fruits are known to synthesis auxin, and recent studies in tomato indicate that auxin is exported out of the fruit and into the peduncles. Proteomic analysis from our studies (see above) indicate auxin signaling is associated with shoots bearing fruit. This year we will perform the following experiments. (1) Determine the proteomic profiles for the phloem sap derived from ON and OFF shoots. We need to repeat this to rule out the possibility that the environment played a role in the accumulation of auxin signaling proteins in the phloem sap of ON shoots. (2) We will determine if crop load alters the PATS system in Arabidopsis. (3) If the PATS system is altered in Arabidopsis in response to fruit load, then we will examine the PATS system in ON and OFF shoots, before and during floral induction.
Impacts
In collaboration with Rueben Hofshi an Advisor for Farm ACW, we obtained a plot of 30 Hass avocado trees at ACW. In a randomized plot, we removed the mature fruit and developing flowers from 15 trees in March 2010 to create our OFF trees. In the remaining 15 trees, fruit was harvested and allowed flowers set fruit to create the ON trees. Phloem sap was collected from the leaves of OFF trees as well as from the shoots bearing fruits in the ON trees. Using a pressure bomb, we collect phloem sap from developing fruits. Proteins were extracted and subjected to proteome analysis Proteomic Core Facility at UCR. Dr. Songqin Pan, the Proteomics Academic Coordinator, assisted in the Proteomic analysis. Proteomic analysis revealed that the phloem derived from the fruits was low in protein content and complexity. Based on these results, we feel that fruits are not likely to produce mobile proteins that move to the shoot apex via the phloem to reduce the reproductive potential of the shoot. However, comparative proteomics analysis of phloem from ON and OFF leaves displayed unique profiles. Of interest, the ON sample contained peptides similar to proteins involved in ubquitination and auxin signaling. Based on these results, I have developed a model of how fruits alter the growth and development of the shoot and how we will implement in the next years research is summarized in the project modifications (see below). AES funding was crucial to help cover part of the salary for Shruti Lal, a graduate student who performed the bioinformatics with the peptides. In addition, AES funding covered the costs for proteomic analysis and supplies.
Publications
- No publications reported this period
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Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: A central goal for plant breeders is to develop new methods and technologies that allow growers and farmers to increase crop productivity and yields. Most of the agricultural foods and products are derived from developmental processes that occur during flowering. A majority of agricultural tree crops produce fruits and seeds in "on" and "off" years, which is referred as alternate bearing. Avocado (Persea americana) production is a multi-million dollar industry in the United States and California produces 90% of the avocados in the U.S. (www.avocado.org). Due to the alternate bearing habit of avocado, fruit production declines in the "off" years, which results in millions of dollars of lost revenue and creates a monetary strain on the growers in California. Recent studies in avocado indicate that the developing fruits produce a mobile systemic signal that alters the identity and activity of the trees shoot apical meristems to prevent flowering. This proposal is aimed at obtaining a global proteomic map from vascular sap (phloem and xylem) in "on" and "off" trees in order to identify mobile proteins that function to inhibit flowering and/or promote proliferative arrest in the reproductive shoots. We refer to these types of proteins as anti-flowering (AF). AF genes will be cloned from avocado and transformed into rapid cycling annual plants to determine if ectopic AF gene expression represses flowering. Once AF proteins are characterized, we will analyze these proteins as well as FLOWERING LOCUS T (FT), a universal flower promoting protein, to determine the relationship and the role of these opposite functioning proteins in alternate bearing in avocado as well as other fruit tree crops. The long-term goal of our research is develop breeding programs to reduce the levels and/or activities of AF proteins so that breeders and growers can control alternate bearing in fruit trees crop to improve efficiency and enhance yields in the "off" years of plant development. Outputs: Outputs 1: We determined the proteomic profiles for vascular sap proteins in "on' and "off" plants. Comparative proteomics was used to determine the proteins that are specifically enriched in the vascular sap of "off" trees. In addition, proteomics was aimed at understanding the biochemical differences between the vascular sap in "on" and "off" trees. Output 2 and 3: Outputs 2 and 3 will determine the function of AF proteins using a rapid cycling plant and correlate alternate bearing with the presence or absence of AF protein and gene expression. Based on the results for Objective 1, we will modify our experimental design in order to complete this part of the proposal. Results from Output 1 have been presented to Dr. Mary Lu Arpaia and Reuben Hofshi, Advisor for the ACW avocado ranch. On June 15th, we will present our findings to the California Avocado Commission at UCR. PARTICIPANTS: This project is collaborative project between my laboratory, Dr. Mary Lu Arpaia, CE Specialist Horticulturist at UCR and Reuben Hofshi, Advisor at the ACW Avocado Ranch in Fallbrook, CA. A Junior Specialist working for Dr. Arpaia, Rodrigo Iturrieta, and a post-doctoral researcher, Dr. Vanitha Ramachandran, were valuable contributors to this project. Mr. Iturrieta received his Masters Degree in Avocado Tree Physiology from the Universidad Catholica Valparasio in Chile. To complement Dr. Iturrieta's background in plant physiology, Mr. Iturrieta has received training in developmental biology, biochemistry and proteomic analysis, while working on this project. Mr. Iturrieta has applied to graduate school, in the Department of Botany and Plant Sciences at UCR. If accepted, he will continue the research on this project gaining molecular experience as part of my collaboration with Dr. Arpaia. Prior to joining my laboratory, Dr. Ramachandran received extensive training in plant virology and microRNA metabolism in plants. In this project, Dr. Ramachandran, received training in proteomics and crop tree development. Reuben Hofshi provided us with avocado trees at the ACW avocado ranch to conduct the experiment. Mr. Mauricio Tapa, Farm Manager for ACW maintained the trees and he has been a valuable consultant on determining floral bud set in the trees. Lastly, an undergraduate in my laboratory, Dr. Nolan Ung, helped collect samples. TARGET AUDIENCES: Our target audience includes growers, farm advisors and mangers as well as plant developmental biologists. Other target audiences include the California Avocado Commission and as well as federal funding agencies. PROJECT MODIFICATIONS: As described above, we obtained a plot of 20 avocado tress all bearing fruit. In order to reset the flowering cycle, we removed the fruits from ten of the trees on June 20th. After removing the fruit, these trees underwent a strong vegetative flush producing leafy shoots. The architecture of the "fruitless" tress was quite distinct from the trees containing the fruit. At the molecular level, distinct differences in the vascular sap proteomic profiles were apparent between the "on" and "off" trees. However, we were unable to detect peptides that match FT in the "on" samples, which should be present in the vascular sap during flowering. Moreover, the shoots of these trees produced few floral buds. Therefore, we conclude that we did not remove the fruit early enough to allow the flowering cycle to reset. This year, we have started removing any flowers produced in "on" population of trees starting in February. The early removal of the flowers should be sufficient to reset the flowering cycle. In addition, we will leave the mature fruit on the other set of trees to extend the alternate bearing cycle another year. Vascular sap will be collected from leaves obtained from these two populations of trees during the later part of summer (end of July through August) when avocado trees undergo the floral transition. In addition, vascular sap will be collected from mature avocados during this time period.
Impacts
In collaboration with Rueben Hofshi an Advisor for the ACW avocado ranch, we obtained a plot of 20 Hass avocado trees at ACW, all of which were bearing fruit. In attempt to reset the flowering, fruits were removed from ten of the trees on June 20th. In the first objective, we had to develop a procedure to isolate proteins from avocado vascular sap. This proved to be quite challenging in that avocado vascular sap is rich in sugars and other metabolites. In the end, we derived an effective method for isolating proteins from leaf and fruit exudates. In our analysis, we collected vascular sap from 6 leaves or fruits, by placing the petioles or peduncles in 20 mls of 1 mM EDTA and 1 mM DTT solution for five hours in a humid chamber. The exudates samples were frozen and dried to a volume of 200 ul. To remove sugars and other metabolites, the exudates were desalted using sephadex G-25 microspin column two times. After determining the appropriate methodology for vascular sap protein isolation, we collected vascular sap from "on" and "off" leaves (flowering vs. alternate bearing, respectively) as well as fruits on August 5th, 26th and September 9th. For the August 26th sample, vascular sap proteins were isolated as described above and digested with trypsin. Proteomic analysis was performed by Dr. Songqin Pan, the Proteomics Academic Coordinator. Proteomic analysis showed that 301 and 268 unique peptides were obtained from "off" and "on" leaves, respectively. In "off" and "on" samples, 44% and 28% of the peptides identified, respectively, matched gene accession numbers in the NCBI database. The putative function of these gene products were involved in signaling, transcription, structural or metabolism. In the "off" and "on" samples, 56% and 28% of the peptides, respectively, did not match any gene accession number. Therefore, the putative function of these proteins is not known. When we compared the two samples, we found that 86% of the peptides (37 peptides) that matched proteins of unknown function were specific to the "off" sample, while 64% (14 peptides) were specific for the "on" sample. In addition, proteomic analysis indicates that the "off" sample contains a significant number of peptides that match proteins involved in ubiquitination, a pattern not found in the "on" sample. The fact that ubiquitination appears to be specific to the "off" vascular sap, indicates that targeted proteolysis may be a crucial element in signaling during alternate bearing. On the other hand, signaling molecules such as kinases, calmodulin as well as 14-3-3 proteins appear to specific to the "on" samples. Interestingly, a higher percentage of peptides matching proteins involved in metabolism were identified in the "on" vs. the "off" sample. AES funding was crucial to help pay the salary of a Dr. Vanitha Ramachandran, a post-doctoral researcher, and cover the costs for proteomic analysis and supplies.
Publications
- No publications reported this period
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