Nonsymbiotic Hemoglobin-2 Leads to an Elevated Energy State and to a Combined Increase in Polyunsaturated Fatty Acids and Total Oil Content When Overexpressed in Developing Seeds of Transgenic Arabidopsis Plants

Abstract

Nonsymbiotic hemoglobins are ubiquitously expressed in plants and divided into two different classes based on gene expression pattern and oxygen-binding properties. Most of the published research has been on the function of class 1 hemoglobins. To investigate the role of class 2 hemoglobins, transgenic Arabidopsis (Arabidopsis thaliana) plants were generated overexpressing Arabidopsis hemoglobin-2 (AHb2) under the control of a seed-specific promoter. Overexpression of AHb2 led to a 40% increase in the total fatty acid content of developing and mature seeds in three subsequent generations. This was mainly due to an increase in the polyunsaturated C18:2 (v-6) linoleic and C18:3 (v-3) a-linolenic acids. Moreover, AHb2 overexpression led to an increase in the C18:2/C18:1 and C18:3/C18:2 ratios as well as in the C18:3 content in mol % of total fatty acids and in the unsaturation/saturation index of total seed lipids. The increase in fatty acid content was mainly due to a stimulation of the rate of triacylglycerol synthesis, which was attributable to a 3-fold higher energy state and a 2-fold higher sucrose content of the seeds. Under low external oxygen, AHb2 overexpression maintained an up to 5-fold higher energy state and prevented fermentation. This is consistent with AHb2 overexpression results in improved oxygen availability within developing seeds. In contrast to this, overexpression of class 1 hemoglobin did not lead to any significant increase in the metabolic performance of the seeds. These results provide evidence for a specific function of class 2 hemoglobin in seed oil production and in promoting the accumulation of polyunsaturated fatty acids by facilitating oxygen supply in developing seeds.

Compartmentation of Triacylglycerol Accumulation in Plants

“…Other work recently pointed to additional factors that can influence FA supply from plastids for TAG synthesis. Overexpression of an Arabidopsis hemoglobin isoform appeared to improve oxygenation of developing seeds and the energetics of carbohydrate conversion to FAs (20). As a result, these transgenic plants were reported to have a 40% increase in total seed FAs compared with wild-type plants.…”

Oil and Protein Accumulation in Developing Seeds Is Influenced by the Expression of a Cytosolic Pyrophosphatase in Arabidopsis

“…For example, most transgenic approaches to increase the oil content of seed are composed either of attempts to increase "pull" toward oil biosynthesis through the increased expression of genes controlling plastidic fatty acid biosynthesis or cytosolic glycerolipid assembly (Roesler et al, 1997;Zou et al, 1997;Jako et al, 2001;Vigeolas et al, 2007;Zheng et al, 2008) or by increasing metabolic "push" in this direction through the overexpression of developmental or global metabolic regulators that enhance the expression of genes encoding enzymes of glycolysis and fatty acid biosynthesis (Shen et al, 2010;Pouvreau et al, 2011;Tan et al, 2011). Notable exceptions include the seed-specific expression of class 2 hemoglobins to address oxygen-limiting conditions during the seed maturation phase (Vigeolas et al, 2011).…”

Hypoxic survival requires a 2-on-2 hemoglobin in a process involving nitric oxide

“…The phylogenetic complexity of Hbs is equaled by the number of reactions catalyzed by the hemoproteins (4). These include O 2 transport and storage also in nonanimal organisms (5,6) as well as sulfide transport and the detoxification of nitric oxide (NO), nitrite, and peroxides (4). As most Hbs are able to catalyze several or all of the possible reactions in vitro, biochemical studies are not sufficient to explain Hb functions in vivo (7).…”