biochemical changes and carbon supply in linseed developing embryos

8/7/2019 Biochemical changes and carbon supply in linseed developing embryos

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BIOCHEMICAL CHANGES AND CARBON SUPPLY IN LINSEED

DEVELOPING EMBRYOS

S. TROUFFLARD

1

, I. PROST

2

, B. THOMASSET

3

, J-N. BARBOTIN

1

, A. ROSCHER

1

, S. RAWSTHORNE

4

,J-C. PORTAIS5

1- Universit de Picardie Jules Verne, UMR-CNRS 6022, 33, rue Saint-Leu, F-80039 Amiens Cedex, France

2- Universit Paul Sabatier, UMR-CNRS 5546, 24, Chemin de Borde-Rouge, 31326 Castanet Tolosan, France

3- Universit Technologique de Compigne, UMR-CNRS 6022, 60203 Compigne, France

4- John Innes centre, Norwich Research Park, Colney, Norwich, NR4 7UH, United Kingdom

5- Laboratoire Biotechnologie-Bioprocds, UMR-CNRS 5504-UR INRA 792, 135 Avenue de Rangueil, 31077

Toulouse, France

Introduction

Plant storage reserves are localised in seeds. The composition of these reserves depends on both the plant species

and the seeds developmental stage. Developing oilseed embryos are known to accumulate mainly oil, as well as

starch and proteins. Metabolic aspects of storage product accumulation have already been studied in oilseed rape

(3, 6). It has been shown that according to the developmental stage of the embryos, carbon flow into lipids and

the composition of storage products change. We initiated the same kind of approach in linseed. Some

information on fatty acid synthesis in linseed has been reported (2), but the metabolic basis of storage product

accumulation in linseed is still not well understood. Preliminary investigations on linseed are presented here and

include (i) measurement of the temporal pattern of storage product accumulation in linseed embryos, (ii) a study

of the nature of carbon substrates that might be implicated in fatty acid synthesis , and (iii) preliminary results of

the influence of light on this synthesis.

Materials and methods

Plant material

Linseed plants were grown in a greenhouse at 18C under a 16 hour photoperiod in daylight supplemented by

sodium light. The age of the embryos was determined by tagging each new flower every day. The age of the

embryos is then expressed as days after flowering (DAF). Embryos were harvested between 10 and 50 DAF to

determine the protein, lipid and starch content. Starch content was determined enzymatically, protein content

was estimated using a dye-binding method (1) and lipid content was measured by pulsed NMR.

Plastid isolation

Plastid preparation was as described by Fox et al. (5). Recovery of plastids from initial homogenates and their

latency were expressed according to the activity of NADP-glyceraldehyde phosphate dehydrogenase (NADP-


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GAPDH) or alkaline pyrophosphatase (AP). Plastid feeding experiments and measurement of incorporation of

carbon into fatty acids and starch were performed according to Eastmond and Rawsthorne (3). Except where

described, all feeding experiments were carried out under very low intensity room lighting in opaque eppendorf

tubes placed in sample racks. For feeding experiments with light, embryos aged from 18 to 25 DAF were used to

do the plastid preparations and incubations were carried out in tubes exposed to an artificial light source (500 Wtungsten/halogen lamp set to provide 300 umoles.m-2.s-1 of photosynthetically active radiation).

Chemicals

Chemicals and reagents were as described in Eastmond and Rawsthorne (3). Radiolabelled substrates were

purchased from NEN Life Science products, (Houndslow, UK).

Results and Discussion

Storage product accumulation

The temporal pattern of storage product accumulation in linseed embryos was determined (Fig 1). The oil

content rises rapidly from the early stage of development to the mid-to- late stage of development (15 to 30 DAF)

while protein accumulation starts later and occurs at a rate of about half that of the oil. Both storage products

reach their maximum at about 30 DAF. There was very little starch accumulation measurable in linseed embryos,

with the maximum value that we report here representing only one twentieth of that in rapeseed (3). Apart from

this difference in starch content the oil and protein accumulation profiles for linseed and rapeseed are similar,

although relative protein content is 2.5 times greater in the former.

Figure 1: Storage product accumulation (lipids, proteins and starch) during the development of linseed embryos

Days after flowering

10 15 20 25 30 35 40 45

ProteinandLipid(mg/embryo)

0.0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

2.0

Starch(mg/embryo)

0.00

0.01

0.02

0.03

0.04

0.05

Protein

StarchLipid


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Carbon supply for storage product accumulation

Feeding experiments on isolated linseed plastids were done to study plastidial capacity for the import/use of

exogenous substrates (lipid and starch precursors), and to then compare these rates to those of lipid accumulationin planta. Plastids were prepared from embryos at between 18 and 24 DAF when lipid accumulation is both

rapid and increases linearly (Fig. 1). Of the possible substrates which may be a source of carbon for starch or

fatty acid synthesis , we supplied 14C-labelled malate, acetate, pyruvate and glucose-6-phosphate (Glc-6-P). The

results were expressed according to the activity of the plastidial enzyme marker: NADP-GAPDH (Table 1).

Table 1: Incorporation of14

C-substrate into fatty acids and starch in linseed plastids. Substrates were supplied at a concentration of 2mM.

Values are the mean +/- SE of measurements made on three separate plastid preparations, or are the mean of two replicated or one single

experiment when the SE is not given.

The calculated rate of starch synthesis from Glc-6-P by intact plastids is about 93 times the in planta rate. Given

the low rate of starch accumulation in whole linseed embryos (Fig. 1), this result suggests that there might be

substantial starch turn -over during embryo development.

Of the substrates tested for fatty acid synthesis, the highest rate was supported by pyruvate, although even in this

case the rate of fatty acid synthesis in vitro accounted only for 10% to the in planta rate. Compared to pyruvate,

other metabolites and especially acetate were used only poorly.

To determine if the use of NADP-GAPDH as a means of expressing the rate of fatty acid synthesis leads to

unexpected errors in the comparison of our in vitro data to in planta rates, the feeding results were expressed

using a different plastidial enzyme marker as a reference: alkaline pyrophosphatase. The recovery and the

latency values obtained from using this enzyme were slightly different from those obtained with NADP-GAPDH

(Table 2). Even when the rate of fatty acid synthesis was expressed per unit of alkaline pyrophosphatase, the

calculated in vitro rate was still low compared to the in planta rate. However, it is intriguing that different

plastidial marker enzymes result in different plastid recovery rates. Also, despite the photosynthetic potential

(see below), no significant light fixation of CO2 could be measured.

Substrates for fatty acid

synthesis

Calculated in vitro rate

nmoles of acetate.h-1.embryo

-1

Estimated in planta rate

nmoles of acetate.h-1.embryo

-1

in vitro/in planta

%

[1-14

C] Glc-6-P 3.2 0.92 2.7

[2-14C] pyruvate 11.8 1.17 10.1

[U-14

C] malate 2.8 0.48 166 1.2

[1-14

C] acetate 1.2 0.51 1

Bicarbonate (ATP,light) 0.16 0.14

Substrates for starch synthesis Calculated in vitro rate

nmoles of hexose.h-1.embryo

-1

Estimated in planta rate

nmoles of hexose.h-1.embryo

-1

[1-14

C] Glc-6-P 3.7 0.066 9307


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Table 2: Activity of two plastidial marker enzymes, NADP -dependent glyceraldehydes 3-phosphate dehydrogenase (NADP-GAPDH) and

alkaline pyrophosphatase (AP), in two independent plastid preparations and in the whole embryos from which the plastids were derived.

NADPH-GAPDH AP

Activity in whole embryos(unit of enzyme

activity/embryo)

Recovery % Activity in whole embryos(unit of enzyme

activity/embryo)

Recovery %

0.011 11.7 0.029 5.8

0.031 4.7 0.025 5.8

Influence of light and substrate concentration on fatty acid synthesis

Developing linseed embryos are chlorophyllous and light has been shown to influence fatty acid synthesis from

acetate by isolated plastids (2). In order to investigate the photosynthetic nature of the developing embryos in

more detail, transmission electron microscopy images were made at different stages of development (Fig 3).

Figure 3: Transmission electron microscopy images at different stages of linseed embryo development.

Chloroplast-like organelles with granal stacks are visible at the earliest stage of development that we studied (14

DAF). The mid-to-late and late stages of development reveal a progressive disappearance of grana and a

significant appearance of lipidic vesicules. As pyruvate was utilised much more effectively than acetate as a

substrate for fatty acid synthesis by the isolated linseed plastids we investigated the effect of light on pyruvate

metabolism. Light enhanced the rate of fatty acid synthesis by a factor of 1.4 (Fig. 4), providing further support

to the hypothesis that light energy can contribute to providing the necessary ATP / reducing power to drive fatty

acid synthesis in the chlorophyllous embryos of linseed (2). The rate of fatty acid synthesis from pyruvate

increased with increasing pyruvate concentration, whether incubations were illuminated or not (Fig. 4). This lack

of saturation with respect to substrate concentration is similar to that seen for pyruvate utilisation by plastids

isolated from castor endosperm (4), and unlike the saturatable activity seen for plastids from rapeseed embryos

(3).


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Figure 4: Influence of light on fatty acid synthesis from pyruvate by isolated plastids

Conclusion

We conclude that (i) starch synthesis and turnover may be important in linseed embryos, despite low levels of

starch accumulation, (ii) that under the incubation conditions described pyruvate is the most effective substrate

for fatty acid synthesis in comparison to a range of other well-characterised potential substrates, and (iii) that

light energy can contribute to driving fatty acid synthesis although whether this occurs through the synthesis of

ATP, NADPH or both is not clear. That the in vitro rate of fatty acid synthesis gave, at best, about 10% of the

expected in vivo rate is not caused by too low a substrate concentration, by lack of light energy, nor by errors

caused by choice of plastidial marker enzyme in making these calculations. It is possible that in our incubation

conditions, NADPH and NADH are not produced endogenously in quantities that are required for fatty acid

synthesis. Studying fatty acid synthesis from substrates such as pyruvate, in the presence of Glc-6-P, would

address whether the plastidial oxidative pentose phosphate pathway could play a role in this provision (3,7).

References

1) Bradford , M.M. ( 1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of

protein-dye binding. Anal Biochem 72, 248-54.

2) Browse, J., Slack, C.R. (1985) Fatty-acid synthesis in plastids from maturing safflower and linseed cotyledons. Planta 166, 74-80.

3) Eastmond, P.J., Rawsthorne, S. (2000) Coordinate change in carbon partitioning and plastidial metabolism during the development of

oilseed rape embryos. Plant Physiol. 122, 767-774.

4)Eastmond, P.J., Dennis, D.T., Rawsthorne, S. (1997) Evidence that a malate/inorganic phosphate exchange translocator imports carbon

across the leucoplast envelope for fatty acid synthesis in developing castor seed endosperm. Plant Physiol. 114, 851-856.

5) Fox, S., Hill, L.M., Rawsthorne, S. (2000) Inhibition of the glucose-6-phosphate transporter in oilseed rape (Brassica napus L.) plastids

by acyl-CoA thioesters reduces fatty acid synthesis. Biochemical Journal 352, 525-532.

6) Kang, F., Rawsthorne, S, (1994) Starch and fatty acid synthesis in plastids from developing embryos of oilseed rape (Brassica napus L.).

Plant J 6, 795-805.

7) Kang, F., Rawsthorne, S, (1996) Metabolism of glucose-6-phosphate and utilization of multiple metabolites for fatty acid synthesis by

plastids from developing oilseed rape embryos. Planta 199, 321-7.

Rate of fatty acid synthesis

0

2

4

6

8

10

12

1 mM 2 mM 4 mM

concentration of substrate (pyruvate)

nmolesofacetate.e

m

bryo-1.h-1

dark

light

biochemical changes and carbon supply in linseed developing embryos

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