Biology Reference
In-Depth Information
fixation of 30-50% of the inorganic carbon from the atmosphere (
Longhurst,
Sathyendranath, Platt, & Caverhill, 1995
). Many microalgal species are exceedingly
rich in oil (
Hu et al., 2008
), which has been widely regarded as potential feedstocks
for the production of biodiesel as a renewable alternative to petroleum fuels (
Wijffels
& Barbosa, 2010
). Realizing this potential, however, demands in-depth knowledge
of algal biology in general, and oil biosynthesis and storage in particular.
The unicellular green alga
Chlamydomonas reinhardtii
is a well-established pho-
tosynthetic model organism for the study of many aspects of biological processes
(
Harris, 2001
) including lipid metabolism (
Liu & Benning, 2012; Moellering,
Miller, & Benning, 2009; Riekhof & Benning, 2009
). Like many other microalgae
(
Hu et al., 2008; Wijffels & Barbosa, 2010
),
Chlamydomonas
cells accumulate oil in
oil droplets under stress conditions such as N deprivation or high salinity (
Goodson,
Roth, Wang, & Goodenough, 2011; Siaut et al., 2011
). It is now well recognized that
oil droplets are dynamic organelles that play crucial roles in cellular energy homeo-
stasis and lipid metabolism and trafficking, rather than inert carbon and energy stores
(
Beller et al., 2008; Guo et al., 2008; Martin & Parton, 2006; Olofsson et al., 2009
).
The current model of oil body biogenesis favors formation of oil droplets through
budding from the endoplasmic reticulum (ER) (
Thiele & Spandl, 2008; Walther
& Farese, 2009
). According to this hypothesis, oil droplets originate from specialized
ER subdomains enriched with enzymes involved in oil biosynthesis. Because the
newly formed oils in these ER domains are unable to integrate into membrane bila-
yers due to lack of polar head groups, they accumulate in the hydrophobic region
between the two leaflets of the ER membrane leading to swelling of the membrane
bilayer and eventually the budding of growing oil bodies from ER into the cytosol.
Alternative hypotheses propose the formation of oil droplets at the ER through bi-
layer excision or vesicular budding (
Walther & Farese, 2009
). Despite some recent
efforts, direct evidence supporting any of these models is still lacking. Many ques-
tions remain regarding the mechanism and machinery involved, the exact site of ini-
tiation, and growth of oil droplets as well as how oil droplet proteins are targeted into
oil droplets. In addition, the ontogeny of oil droplets is likely to vary among different
organisms and even between different species of the same organism. For example,
recent data from our own (
Fan, Andre, & Xu, 2011
) and other (
Goodson et al., 2011
)
laboratories indicated that oil droplets in
Chlamydomonas
likely originate from the
envelope membranes of chloroplasts, instead of ER. In this chapter, we will present a
detailed description of procedures that can be used to analyze the oil droplets in
microalgae and to isolate genetic mutants defective in oil droplet biogenesis and
oil accumulation in the model alga
C. reinhardtii
.
5.1
NILE RED AS A PROBE FOR OIL DROPLETS
As a lipid-soluble fluorescent dye, Nile red is a lipid probe due to its specific in-
teraction with hydrophobic molecules (
Greenspan, Mayer, & Fowler, 1985
). Over
the past two decades, Nile red has been widely employed for detection of oil
