100 Years of Breeding Guayule - Industrial Crops: Breeding for BioEnergy and Bioproducts - page 357

Moreover, seasonal expression analysis of known and putative rubber biosynthesis-
related genes from field-grown guayule shrubs found no cold induction, except
perhaps for CPT in which case a sudden increase in ambient temperature at night
10 days before harvest was associated with the highest expression level. Addition-
ally, no positive correlation of expression levels for all genes with rubber transfer-
ase activity was found. Ponciano et al. [ 43 ] concluded that either [ 1 ] gene
expression is not controlling the enzymatic activity of the rubber transferase
complex, but instead posttranslational modifications are the point of control, or
[ 2 ] proteins encoding the genes analyzed are not those regulating rubber biosyn-
thesis, thus the critical member(s) of the rubber transferase complex are yet to be
identified.
Two studies have been conducted to evaluate the performance of genetically
engineered lines. In the first study, Veatch et al. [ 44 ] overexpressed allylic pyro-
phosphate initiators from the isoprenoid pathway, including farnesyl pyrophosphate
(FPP) in guayule. They found natural rubber production was not significantly
altered, but there was enhanced terpenoid resin production in the lines. Only
empty vector controls were used in this study.
In a later study, Dong et al. [ 45 ] evaluated transgenic lines involving the enzyme
3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR). This study included
two types of controls not included in the previous study by Veatch et al. [ 44 ], first
was a tissue culture control and the second was a non-transformed control derived
from seed in addition to the empty vector control. HMGR catalyzes the irreversible
conversion of 3-hydroxy-3-methylglutaryl-CoA to mevalonate, a precursor of IPP.
This enzyme is considered a key regulatory enzyme of MEV carbon flux in
mammals and in microbial systems [ 46 ] and possibly in plants as strategic to
increased isoprenoid production.
Dong et al. [ 45 ] found insertion of the HMGR gene into guayule produced a
higher rubber content phenotype in tissue culture for one line of modified plants and
produced a dwarf phenotype in the field for that same line. Three other lines did not
yield differentiable phenotypes. In order to resolve the discrepancies, gene expres-
sion was measured by quantitative real-time PCR analysis for both tissue culture
and field-grown plant tissues. Results confirm higher expression of the HGMR gene
in modified plants than in control plants. The relative differences in Ct (cycle
threshold) values for all tissue culture plants were lower than that of the empty
vector control or a non-transformed control in all cases. The reduction in Ct value
was even more pronounced for field-grown plants versus the respective controls.
However, in no case did the expression vary significantly between lines for which
phenotype differences were observed. Field evaluation of plants confirmed trans-
genic expression but did not validate metabolite accumulation, probably due to the
overwhelming influence of the environment over many months in the temperature
extremes typically experienced in Arizona. Survival during regrowth of the
transformed lines was significantly improved for HMGR overexpressing plants,
suggesting enhanced carbon flux to important secondary isoprenoid metabolites,
such as growth phytohormones.