Recipient Organization
UNIVERSITY OF ALABAMA IN HUNTSVILLE
301 SPARKMAN DRIVE
HUNTSVILLE,AL 35899
Performing Department
(N/A)
Non Technical Summary
In trees, secondary vascular tissues made up xylem vessels and supporting ground tissues form wood which is the major source of biomass for energy and raw materials for the pulp and paper industry. Relatively little is known about how these two complex vascular tissues differentiate from the meristematic cells of the vascular cambium. The focus of this proposal is to identify genes that are likely to be important in the differentiation and development of secondary vascular tissues and characterize their function through expression studies and analysis of mutations in transgenic plants. The purpose of this project is to identify and charcaterize homeotic genes that control and regulate the genes involved in wood formation ( secondary vascular tissues) in trees.
Animal Health Component
40%
Research Effort Categories
Basic
60%
Applied
40%
Developmental
(N/A)
Goals / Objectives
(1) identify and clone full length MADS-box cDNAs from cDNA libraries prepared from secondary vascular tissues (2) characterize the expression patterns of these MADS-box genes during wood formation and (3) select the best candidate MADS-box cDNA to begin construction of expression vectors to analyze the function of these MADS-box genes by altering their level of expression in transgenic aspen. Construction of transgenic aspen plants and analysis for altered expression of vascular specific MADS-box genes will take well beyond the proposed three-year project period.
Project Methods
We have accomplished the first objective by cloning and characterizing several cDNAs from developing vascular tissues of aspen. From those cDNAs we have identified and cloned the full-length clone of PTM5 using a cDNA library prepared from developing vascular tissues of aspen. The PTM5 cDNA is approximately 1000 bp long and includes ~40 bp of 5'-UTR and ~148 bp of 3'-UTR. PTM5 showed ~ 60% homology at the amino acid level to AGL20 MADS-box genes from Arabidopsis and thus seems to be a new class of MADS-box gene. We have completed the second objective also. Analysis by RT-PCR followed by southern blot using specific 3'-end probes from PTM5 have shown that this gene is expressed very early in spring season in the developing vascular tissue. We have now confirmed that PTM5 is vascular specific and is expressed during May and June and its levels of expression were non-detectable by July. We have optimized the protocols for the temporal and spatial expression patterns (in
situ) of PTM5 during the seasonal growth of woody tissues. The in situ data clearly confirms the vascular-specific nature of PTM5 and that PTM5 expression is limited to few layers of vascular cambium during secondary vascular tissue development in the spring. This is the first MADS-box gene identified that is exclusively expressed in developing primary and secondary vascular tissues. Thus, it seems PTM5 is very useful homeotic gene possibly involved in controlling the spring wood development. We have made significant progress on the third objective. We have completed construction of expression vectors for protein expression of PTM5 and also vectors for testing the transgenic expression of PTM5 in aspen. Because we were able to hire the postdoc only after a year of original project start date, we request for a no cost extension of our project till the end of this year. In the remaining period, we plan to initiate the transformation of aspen with over expression constructs of PTM5. We
plan to continue the work in the next project period to specifically determine the functional genomics of PTM5 in heterologous systems such as Arabidopsis and tobacco and homologous system aspen to study its effects on wood formation.