Phosphatidylglycerol Composition Is Central to Chilling Damage in the Arabidopsis fab1 Mutant

The Arabidopsis (Arabidopsis thaliana) fatty acid biosynthesis1 (fab1) mutant has increased levels of the saturated fatty acid 16:0, resulting from decreased activity of 3-ketoacyl-ACP synthase II. In fab1 leaves, phosphatidylglycerol, the major chloroplast phospholipid, contains >40% high-melting-point molecular species (HMP-PG; molecules that contain only 16:0, 16:1-trans, and 18:0 fatty acids)—a trait associated with chilling-sensitive plants—compared with <10% in wild-type Arabidopsis. Although they do not exhibit short-term chilling sensitivity when exposed to low temperatures (2°C to 6°C) for long periods, fab1 plants do suffer collapse of photosynthesis, degradation of chloroplasts, and eventually death. To test the relevance of HMP-PG to the fab1 phenotype, we used transgenic 16:0 desaturases targeted to the endoplasmic reticulum and the chloroplast to lower 16:0 in leaf lipids of fab1 plants. We produced two lines that had very similar lipid compositions except that one, ER-FAT5, contained high HMP-PG, similar to the fab1 parent, while the second, TP-DES9*, contained <10% HMP-PG, similar to the wild type. TP-DES9* plants, but not ER-FAT5 plants, showed strong recovery and growth following 75 d at 2°C, demonstrating the role of HMP-PG in low-temperature damage and death in fab1, and in chilling-sensitive plants more broadly.

In higher plants, the chloroplast membranes that host the light harvesting and electron transport processes of photosynthesis have a characteristically high number of double bonds in the glycerolipid acyl chains. Only ∼10% of the fatty acids that compose the hydrophobic core of the thylakoid bilayer lack double bonds altogether, whereas >80% are polyunsaturated, having two or three double bonds (Ohlrogge et al., 2015). The photosynthetic light reactions produce reactive oxygen species as by-products, and these can degrade polyunsaturated fatty acids, so it is assumed that highly unsaturated membranes are required to support photosynthesis (McConn and Browse, 1998).The glycerolipids in chloroplast membranes are synthesized by two separate pathways. (Browse et al., 1986; Ohlrogge and Browse, 1995). Synthesis de novo of fatty acids takes place in the stroma of chloroplasts, producing 16:0 esterified to acyl carrier protein (ACP). A large proportion of this 16:0-ACP is elongated by 3-keto-acyl-ACP synthase II (KASII) to 18:0-ACP, which is in turn desaturated by stearoyl ACP desaturase to produce 18:1-ACP (Lindqvist et al., 1996; Carlsson et al., 2002). The fatty acids from 16:0-ACP and 18:1-ACP may be used within the chloroplast in the prokaryotic pathway (Kunst et al., 1988; Kim and Huang, 2004) to produce phosphatidic acid (PA). Some of this PA intermediate is used for synthesis of phosphatidylglycerol (PG; Ohlrogge and Browse, 1995; Wada and Murata, 2007), which is the only chloroplast glycerolipid that is produced solely by the prokaryotic pathway. In some plants, including Arabidopsis (Arabidopsis thaliana), PA is also converted to diacylglycerol (DAG), which is the precursor for the synthesis of the other chloroplast glycerolipids, monogalactosyldiacylglycerol (MGD), digalactosyldiacylglycerol (DGD), and sulfoquinovosyldiacylglycerol (SQD; Browse et al., 1986; Ohlrogge and Browse, 1995; Ohlrogge et al., 2015).The second route for chloroplast glycerolipid synthesis, the eukaryotic pathway, begins with export of 16:0 and 18:1 from the chloroplast as CoA thioesters. (Li et al., 2015). In the endoplasmic reticulum (ER), these fatty acids are rapidly incorporated into phosphatidylcholine (PC) by acyl exchange (Bates et al., 2007), and are also used (via PA and DAG intermediates) for the synthesis of all the phospholipids of the extrachloroplast membranes of the cell (Ohlrogge et al., 2015). In addition however, the DAG moiety of PC can be returned to the chloroplast and contribute to the production of MGD, DGD, and SQD required for thylakoid synthesis (Benning, 2009; Roston et al., 2012). The ER-to-chloroplast flux of lipid is reversible to some extent (Browse et al., 1989, 1993).With the exception of the first Δ9 double bond in 18:1-ACP, all the double bonds in the acyl chains are introduced after the initial synthesis of glycerolipid molecules. In Arabidopsis, this involves the action of seven fatty acid desaturases that are integral membrane proteins in the chloroplast and ER (Ohlrogge and Browse, 1995; Wallis and Browse, 2010). Characterization of Arabidopsis fatty acid desaturation (fad) mutants deficient in one or more of these desaturases has shown that the high level of thylakoid unsaturation is essential to photosynthetic function (Murakami et al., 2000; Routaboul et al., 2000). For example, fad2 fad6 double-mutant plants are unable to synthesize polyunsaturated fatty acids and cannot grow autotrophically; however, when grown on Suc as a carbon source, the double mutants are robust plants showing strong leaf and root development (McConn and Browse, 1998). These results indicate that the vast majority of receptor-mediated and transport-related membrane functions required to sustain the organism and induce proper development are adequately supported in the absence of polyunsaturated lipids; photosynthesis is the one process that requires high levels of polyunsaturation. Mutants with smaller changes in unsaturation are often similar to the wild type under typical growth-chamber conditions and reveal their phenotypes only under more extreme conditions (Wallis and Browse, 2002, 2010). Several mutants grow more slowly and become chlorotic at temperatures in the range 2°C to 10°C (Hugly and Somerville, 1992; Routaboul et al., 2000), indicating a role for fatty acid composition in maintaining photosynthesis at these low temperatures.Like other species native to temperate regions, Arabidopsis is chilling resistant and able to grow at temperatures close to 0°C. By contrast, many tropical and subtropical plant species are chilling sensitive and suffer sharp reductions of photosynthesis and extensive tissue damage after even short exposure to low temperatures. Many of the world’s most important crops, including rice (Oryza sativa), maize (Zea mays), and soybean (Glycine max) are chilling sensitive, so a better understanding of the biochemical and genetic factors contributing to this sensitivity has the potential to enhance sustainable food production (Nishida and Murata, 1996; Iba, 2002; Thakur et al., 2010). One hypothesis proposes that chilling sensitivity is a result of the fatty acid composition of chloroplast PG. It is based on the observation that many chilling-sensitive plants contain >30% of PG molecules with only saturated or trans unsaturated fatty acids—16:0, 18:0, and 16:1-Δ3trans (16:1t)—at both the sn-1 and sn-2 positions of the glycerol backbone, referred to as high-melting-point molecular species (HMP-PG; Murata, 1983; Barkan et al., 2006). This name alludes to the fact that HMP-PG species can induce a phase change from liquid crystalline (typical of biological membranes) to gel phase at temperatures well above 0°C and thereby disrupt membrane and cellular function (Murata and Yamaya, 1984). Chilling-resistant plants have <10% HMP species in chloroplast PG (Murata et al., 1982; Murata, 1983; Roughan, 1985).One perspective on the role of HMP-PG in plant temperature responses has come from our investigations of the fatty acid biosynthesis1 (fab1) mutant of Arabidopsis. In this mutant, a hypomorphic mutation in the gene encoding KASII reduces elongation of 16:0-ACP to 18:0-ACP (Carlsson et al., 2002), producing plants that have increased levels of 16:0 in all membrane glycerolipids (Wu et al., 1994). In particular, fab1 plants contain HMP-PG at levels (∼40% to 50% of total PG) similar to those of many chilling-sensitive plant species (Wu and Browse, 1995). Nevertheless, the fab1 mutant does not show typical symptoms of chilling sensitivity and is unaffected, in comparison to wild-type controls, by a range of chilling treatments that kill chilling-sensitive plants; instead, fab1 plants only show a collapse of photosynthesis after >10 d of exposure to 2°C, with the plants dying after several weeks at low temperature (Wu and Browse, 1995; Wu et al., 1997).We have previously screened for genetic suppressors of the fab1 low-temperature phenotype. Most, though not all, of the suppressor mutations substantially reduce the proportion of saturated fatty acids in PG, consistent with the notion that HMP-PG causes eventual death of fab1 plants in the cold (Barkan et al., 2006; Kim et al.,2010; Gao et al., 2015). However, all the suppressors have additional changes, relative to fab1, in the fatty acid compositions of membrane lipids that prevent a clear linkage between reductions in HMP-PG and improved low-temperature survival.Here, we have taken a new approach to investigating the role of HMP-PG in damage and death of fab1 plants at chilling temperatures by using a 16:0-CoA desaturase from Caenorhabditis elegans, FAT-5 (Watts and Browse, 2000), and a glycerolipid desaturase, DES9*15, derived from a cyanobacterial enzyme by directed evolution (Bai et al., 2016). When expressed in the fab1 mutant background, both the FAT-5 enzyme targeted to the ER and the DES9*15 enzyme targeted to the chloroplast reduced leaf 16:0 to near-wild type levels. The fatty acid compositions of individual leaf lipids in plants of both transgenic lines were very similar, with the sole exception of PG. Plants expressing the FAT-5 desaturase retained high levels of HMP-PG, similar to fab1, while plants expressing the DES9*15 enzyme had HMP-PG lowered to levels close to those of the wild type. Like the fab1 mutant, fab1 plants expressing a 16:0 desaturase in the ER lost photosynthetic function over 28 d of exposure to 2°C and showed little capacity for recovery and growth after longer periods at low temperature. By contrast, plants expressing a 16:0 desaturase targeted to the chloroplast retained substantial photosynthetic function, even after 75 d at 2°C, and were subsequently able to resume growth, flower, and set seed upon return to 22°C. These results provide the most direct evidence yet that high levels of HMP-PG cause gradual loss of photosynthesis and eventual death of plants at chilling temperatures.     

GENES MEET GEOLOGY: FISH PHYLOGEOGRAPHIC PATTERN REFLECTS ANCIENT, RATHER THAN MODERN, DRAINAGE CONNECTIONS

Abstract We used DNA analysis of the freshwater Galaxias vulgaris complex (Pisces: Galaxiidae) to test a geological hypothesis of drainage evolution in South Island, New Zealand. Geological evidence suggests that the presently north-flowing Nevis River branch of the Clutha/Kawarau River system (Otago) once flowed south into the Nokomai branch of the Mataura system (Southland). The flow reversal is thought to have resulted from fault and fold activity associated with post-Miocene uplift. Mitochondrial DNA sequence data (control region and cytochrome b genes; 76 individuals; maximum divergence 7.1%) corroborate this geomorphological hypothesis: The Nevis River retains a freshwater fish species ( Galaxias gollumoides ; five sites; 10 haplotypes) that is otherwise restricted to Southland (nine sites; 15 haplotypes). There is no indication that the Nevis River lineage of G. gollumoides lives elsewhere in the Clutha/Kawarau system (> 30 sites). Likewise, two widespread Clutha lineages ( G. 'sp' D; G. anomalus–G. pullus ) are apparently absent from the Nevis (> 30 sites). In particular, G. 'sp D' lives throughout much of the Clutha (12 sites, 23 haplotypes), including a tributary of the Kawarau, but is absent from the Nevis itself. Conventional molecular clock calibrations (based on a minimum Nevis-Mataura haplotype divergence of 3.0%) indicate that the Nevis flow reversal may have occurred in the early-mid Pleistocene, which is roughly consistent with geological data. The broad phylogeographic structure evident in the Clutha system is consistent with the sedentary nature of nonmigratory galaxiids. Our study reinforces the value of combining biological and geological data for the formulation and testing of historical hypotheses.     

The molecular evolution of vertebrate growth hormones: A pattern of near-stasis interrupted by sustained bursts of rapid change

It has been demonstrated previously that in mammals the evolution of pituitary growth hormone shows an unusual pattern, with an underlying slow rate and at least two sustained bursts of rapid evolution (in the artiodactyls and primates), during which the rate increased at least 25-fold. It is demonstrated here that a similar pattern applies for growth hormone evolution throughout the vertebrates, with a basal rate similar to that seen in mammals, but bursts of rapid evolution in the amphibia and the elasmobranchs, and several bursts in the teleosts. The placental growth-hormone-like proteins of primates show a similar pattern. It is argued that the bursts of evolution seen for growth hormone are a consequence of selection and that this may reflect changes in the functions of the hormone additional to its basic growth-promoting actions.