Identification of a Highly Expressed 3-Hydroxy-3-Methylglutaryl-CoA Reductase Gene in the Root Tissue of Taraxacum kok-saghyz

Kazakh dandelion (Taraxacum kok-saghyz, Tk) is a rubber-producing plant currently being investigated as a source of natural rubber for industrial applications. Like many other isoprenoids, rubber is a downstream product of the mevalonate pathway. The 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR) enzyme catalyzes the conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonic acid, a key regulatory step in the MVA pathway. Such regulated steps provide targets for increases in isoprenoid and rubber contents via genetic engineering to increase enzyme activities. In this study, we identify a TkHMGR1 gene that is highly expressed in the roots of Kazakh dandelion, the main tissue where rubber is synthesized and stored. This finding paves the way for further molecular and genetic studies of the TkHMGR1 gene, and its role in rubber biosynthesis in Tk and other rubber-producing plants.


Introduction
The main source of natural rubber in the world is the rubber tree Hevea (Hevea brasiliensis) grown mainly in Southeast Asia [1] [2].Other rubber-producing plants, such as Kazakh dandelion (formerly Russian dandelion) (Taraxacum kok-saghyz, Tk) and Guayule (Parthenium argentatum) are currently being developed as alternative crop sources of natural rubber [3]- [6].
Constitutive over expression of HMGR genes has proven to be effective in increasing production of selected isoprenoids and end-product sterols in plants [26]- [28].The main organ of rubber synthesis and storage in Tk is the root.To increase rubber biosynthesis in Tk by means of genetic engineering, one approach is to over express a HMGR in the root tissue.Here we report the identification of a root-specific TkHMGR1 through data mining and gene expression studies.

Plant Material
Tk seeds were obtained from the United States Department of Agriculture-Agricultural Research Service National Plant Germplasm System, where they were deposited after collection in Kazakhstan in 2008 (accession No. KAZ08-014, ID W6-35169).Plants were germinated and grown in a greenhouse at temperatures between 28˚C (day) and 18˚C (night), with supplemental metal halide lighting to provide a 15-h-day length (1000 to 1250 µmol m −2 •s −1 ).

Computational Analysis of TkHMGR-Like Expressed Sequences
Hevea HMGR1 (X54659) was used as query for homology searches (tblastn) of a Tk root tissue expression library DNA sequences available at the National Center for Biotechnology Information (NCBI).Seventeen TkHMGR-like partial sequences were found with an average length of 750 bp (Table 1).Eleven of the sequences were homologous to the N-terminus of Hevea HMGR1, three to the C-terminus, and three to the middle portion of the protein (between amino acids 167 -426).The large numbers of N-terminus homology sequences and the low representation of sequences with C-terminus homology suggest a technical problem during construction of the cDNA library.HMGR is known to be present in plants in several isoforms.Without full-length TkHMGR sequence information, it is impossible to determine the number of TkHMGR isoforms.However, spatial (organ specific) and temporal (age specific) expression analysis of these sequences can provide useful information about the presence of tissue-specific isoforms.

Spatial and Temporal Analysis of Tk HMGR-Like Sequences
We aligned all 17 Tk sequences against the Hevea HMGR1 gene (X54659) and designed five sets of PCR primers.Computational analysis of the five primer sets predicted that each would amplify at least one of the following 6 sequences, GO661008, GO662289, GO668051, GO666861, GO665221, and GO670200.PCR amplifications were performed on cDNAs derived from total RNA extracted from leaf, root, and flower tissues of ~11 weeks old Tk plants.All primers sets amplified a product in all tissues (data not shown).However one primer set (Materials and Methods) consistently amplified an abundant product from cDNAs from root tissues.This primer set, herein called TkHMGR1 primers, was used for quantitative expression analysis.Computational analysis of TkHMGR1 primers predicted only one PCR product from only one sequence, GO666861, and, interestingly, no amplification from the cloned TkHMGR sequence (HQ857601).To examine the relationship between rubber accumulation and TkHMGR1 expression, we further quantified TkHMGR1 transcript levels in root and leaf of young (~11 weeks old) and mature (~20 weeks old) Tk plants using qPCR technology.In our lab, rubber extractions from Tk roots typically yield 4 mg/g fresh weight from 12-week-old plants and 10 mg/g fresh weight from 20-week old plants).No rubber was detected in leaf tissues.As shown in Figure 1, we detected 11-fold or 3-fold higher of TkHMGR1 transcript levels in roots than in leaves from young or mature plants, respectively.The results indicated that the expression of TkHMGR1 was higher in root than leaf during the active growth phase of Tk plants.Although functional studies need to be carried out in order to determine the enzymatic activity and substrate specificity of TkHMGR1 in rubber metabolism, it is likely that TkHMGR1 contributes to rubber synthesis in Tk roots based on its expression profile.
Rubber is the end metabolite of the MVA biosynthesis pathway.The amount of rubber in mature root is the result of continuous synthesis and accumulation over the lifespan of the plant.Although we observed a decline in expression of TkHMGR1 in roots when plants became mature (Figure 1), the level of the transcript was probably still high enough to produce active TkHMGR1 enzymes for rubber biosynthesis.It is also possible that TkHMGR1 is a relatively stable protein, and thus supports continuous biosynthesis of rubber over development, leading to the high accumulation of rubber in root.
In summary, we have identified the TkHMGR1 isoform that is highly expressed in Tk root.The C-terminal part of TkHMGR1 sequences are represented by GenBank accession GO666861.The partial sequence provided the basis for the design of isoform-specific primers that can be used to isolate full-length TkHMGR1 and eventually its promoter sequences.The likely role that TkHMGR1 plays in rubber synthesis suggests that its overexpression in guayule or Tk could increase rubber production.In addition, the TkHMGR promoter can be used to drive gene expression for other key enzymes/proteins associated with rubber synthesis in Tk root, such as the genes for IPP synthase, cis-prenyltransferase, rubber elongation factor, etc.Thus, the sequence identified in this study could lead to other valuable tools for genetic engineering of rubber-producing crops.

Figure 1 .
Figure 1.Expression of TkHMGR1 in root and leaf of young (11 week) and mature (20 week) Tk greenhouse plants.Expression levels determined by qPCR are relative to those of 18S ribosomal RNA in the same samples.Each data point represents the mean ± SD of three replicates.

Table 1 .
List of TkHMGR-like sequences showing homology to Hevea HMGR1.