Introduction
16:0 and 18:0 fatty acids are the primary products of de novo fatty acid synthesis in the plastids. Most of
the 18:0 synthesized is immediately desaturated into 18:1. These fatty acids are either used in
chloroplast lipid synthesis via acyl transferases or exported into the endoplasmic reticulum via acyl-ACP
thioesterases. There are two different classes of acyl-ACP thioesterases called FATA and FATB. FATA is
encoded by two genes and was found to have a high activity for 18:1-ACP with a lower activity for
saturated fatty acids. FATB is encoded by one gene and was found to have the highest activity for
saturated fatty acids with some activity for 18:1-ACP. These differences in enzyme specificity determine
the amount and the type of fatty acids that are exported from the plastids.
Previous studies have shown that the primary fatty acid exported from the plastid is 18:1 and from
experiments on in vitro activity, we can predict that FATA is the enzyme responsible for the in vivo levels
of 18:1 that is exported from the plastid. However, the importance of FATB was not as obvious as FATA.
FATB has been shown to have a minor contribution to the total flux of carbon in glycerolipid synthesis,
but since FATA also has some affinity for saturated fatty acids, the authors of this paper wanted to know
what the importance of FATB was and why there is a need for two classes of acyl-ACP thioesterases.
Results
First the authors screened for potential mutants in a population of tDNA-tagged Arabidopsis plants for
FATB gene disruption by performing a PCR analysis on pooled leaf genomic DNA to find a tDNA insertion,
encoding a Basta resistance gene, in the second intron of the gene. They found that the mutant plants
had a slower growth rate and a different fatty acid composition than wild type plants.
To see if the mutation is heritable and how many genes were involved, heterozygous plants were selffertilized and the progeny were screened on Basta to observe the resistant to susceptible ratio. The ratio
was 3:1 as expected and showed that the mutation is in a single nuclear gene.
A co-segregation analysis was performed to determine if the tDNA insertion is the mutation responsible
for the observed phenotypes. Basta resistant plants were genotyped and their fatty acids compositions
were assessed using GC-FID. Plants with a wild type appearance and fatty acid composition were
heterozygous for the tDNA insertion, while plants with a slow growth rate and a mutant fatty acid
composition were homozygous for the insertion. This experiment showed that the tDNA insertion is a
recessive mutation and segregates with the mutant phenotype.
In their complementation analysis, they transfected homozygous mutants with a vector containing wild
type FATB cDNA and Hygromycin B resistance and selected for plants resistant to both Basta and
Hygromycin B. The transformed mutants were found to have wild type fatty acid composition and
normal growth, which showed that a tDNA insertion into the FATB gene is responsible for the mutant
phenotype.
Even though a tDNA insertion into an intron would interrupt normal splicing, some wild type mRNA may
still be made. Correctly spliced mRNA was indeed detected in the mutants through RT-PCR. qPCR with
elF4A1 as an internal control was used to determine how effective the mutation was by evaluating how
much of the normal mRNA was still being made by the mutant plants. They found that the mutant
transcript levels were 150-fold lower than wild type levels. As well, a protein gel blot analysis using antiArabidopsis FATB antibodies was unable to detect FATB protein in the mutant. These experiments
showed that the mutant isolated was an effective mutant.
Mutants were grown to compare their phenotypes to wild type plants. Bolting time was longer and
growth rate was lower in the mutant than in the wild type. Since previous mutants with differences in
fatty acid composition had a wild type phenotype when growing temperature was increased or
decreased, the mutants were grown at 16°C, 22°C, and 36°C. At each of the temperatures, the mutant
had a 50% reduction of fresh weight compared to wild type plants, which showed that the mutant
phenotype was not affected by temperature. When they looked at seeds using a scanning electron
microscope, mutant seeds were found to have a range of deformity from wild type-like to very
deformed with a shriveled seed coat.
Finally, they analyzed the fatty acid compositions of the mutant and wild type plants. The fatty acids
were stripped from their lipids using strong alkaline hydrolysis to evaluate both O-linked (glycerolipids)
and N-linked (sphingolipids) fatty acids. Leaf waxes were also analyzed because these are synthesized
from 16:0 and 18:0 fatty acids. They found that there was a decrease in surface waxes in leaf and
primary stem waxes with a greater decrease in stem waxes.
When the fatty acid composition of sphingolipids was analyzed, they found that there was no significant
decrease in the total proportion of sphingoid bases between wild type and mutant plants. However,
there were changes in the relative abundance of individual sphingoid bases. This showed that
sphingolipids are essential to plant growth and they are maintained at the expense of the fatty acid
composition of other lipids.
If FATB activity is lost, it is possible that FATA activity would be increased to compensate. To test this,
they performed an assay on 16:0 and 18:1 hydrolysis and found that 18:1 hydrolytic activity was the
same in both wild type and mutant plants, which showed that there was no upregulation of FATA.
In FATB mutants, the decrease of saturated fatty acids was only 50%. A double mutant of fatb-ko and
act1 was created to see what percentage of saturated fatty acids comes from the prokaryotic pathway.
This double mutant had a severely impaired growth and had a 70% decrease of 16:0 compared to wild
type plants. From this experiment, we can see that saturated fatty acids are essential to maintaining
growth rate.
Discussion
This paper showed that saturated fatty acids are critical for plant growth and a decrease in only 50% was
able to slow growth rate significantly. A change in growth temperatures was unable to produce a normal
phenotype in the mutant. Sphingolipids were maintained at the expense of other lipids because they are
a critical component of plasma membranes. Even though we now know that FATB is important to plant
growth, we still do not know what the specific functions of saturated fatty acids in sustaining normal
growth are. Some future studies would look at if growth rate is linked to biosynthesis of cell components,
changes in membrane properties or fatty acid synthase, or a combination of all of these.