Ian McGregor1, Shankar Das2, Brian Miki3, Wilf Keller2 and Ping Fu1



1Agriculture and Agri-Food Canada, 107 Science Place, Saskatoon, SK, Canada

S7N 0X2 (mcgregordi@em.agr.ca),

2National Research Council, Plant Biotechnology Institute, 110 Gymnasium Road, Saskatoon, SK,

 Canada  S7N 0W9,

3Eastern Cereal and Oilseed Research Centre, Agriculture Agri-Food Canada, K.W. Neatby Building,

 Ottawa, ON, Canada K1A 0C6




A yeast-derived invertase, driven by the seed-specific napin storage protein promoter, was transformed

 into Brassica napus L. cv Westar canola with overexpression targeted to the cytosol or the apoplast.

 The intent was to precociously enhance chlorophyll clearing in maturing seed and thereby address the

 "green seed problem". Homozygous plants were obtained with up to 12-fold increased expression over

 Westar of soluble acid invertase in the cytosol. Overexpression of invertase targeted to the apoplast led

 to increase in both soluble and insoluble acid invertase. Homozygous plants were obtained with up to

 11-fold increased expression over Westar of total acid invertase. A developmental study with selected

 lines expressing invertase targeted to the apoplast indicated that invertase expression did not deviate

 over the filling phase of seed maturation. Relatively low levels of germination of R1 seed suggested

 that appreciable overexpression of invertase, particularly when targeted to the apoplast, may interfere

 with germination. Developmentally, neither the peak accumulation nor the timing or rate of chlorophyll

 clearing was shown to be influenced by the achieved level of overexpression of apoplastic invertase.



KEYWORDS  Brassica napus, apoplastic invertase, cytosolic invertase, Agrobacterium-mediated

 transformation, seed chlorophyll content, germination





The cotyledons of developing canola (Brassica napus L. and B. rapa L.) embryos are rich in chlorophyll

 up to mid-maturation phase (500 to 800 g­ × g-1 fresh matter), then undergo a rapid programmed loss

 that is usually completed well before the seed is mature (Johnson-Flanagan and Thiagarajah, 1990;

 McGregor, 1991). When chlorophyll is retained in the mature canola seed as the result of an early frost

 or other environmental factors (the "green seed problem") producers experience substantial economic

 losses. Estimates of loss have ranged as high as 50 to 100 million in some years (Underwood, 1995).

 As little as 3%­distinctly green seed (>20 ­g­ × g­-1 fresh matter; >20 ppm chlorophyll) reduces the

 value of the crop. Chlorophyll extracts with the oil during processing (Yuen and Kelly, 1980; Appelqvist,

 1989). Chlorophyll can inhibit the hydrogenation catalyst used for hardening in the manufacture of

 margarine (Abraham and De­Man, 1986). Oils from seed with elevated chlorophyll content are less

 stable, their oxidation resulting in rancidity (Dahléns, 1973). Chlorophyllides and pheophorbides,

 phytol-deficient chlorophyll derivatives produced during processing, may contribute to photosensitive

 dermatitis (Clare, 1955). Although technology exists for the removal of chlorophyll from the oil during

 processing, removal adds to the cost of processing. Development of germplasm with improved ability to

 clear chlorophyll before maturity is a permanent solution to reduce or eliminate green seed.


By varying the time of seeding, it has been shown that chlorophyll clearing in canola seed occurs at a

 relative constant rate (McGregor, 1995). It has also been shown that the timing of chlorophyll clearing

 may shift in relation to seed development (McGregor, 1995). Seeding early to ensure that the seed was

 filling when temperatures were favourable, resulted in chlorophyll clearing occurring well in advance of

 seed moisture loss. Seeding later so that the seed was filling under cooler temperatures, resulted in

 chlorophyll clearing occurring along with the loss of seed moisture. Reduction in the temporal

 separation between chlorophyll clearing and moisture loss would appear to contribute to elevated

 residual chlorophyll content in mature seed. Chlorophyll becomes entrapped when moisture content of

 the seed drops to the point that metabolic processes are curtailed and thus further breakdown of

 chlorophyll can not occur.


Swathing studies have also shown that chlorophyll clearing occurs at a relatively constant rate once

 initiated, and that swathing can advance the time of both chlorophyll clearing and moisture loss

 (McGregor, 1995). If chlorophyll clearing was not underway at the time of swathing, swathing initiated

 the process. The rate of clearing in the swathed crop was comparable to that which would have

 subsequently occurred if the crop had been left standing. Thus, varying environmental conditions

 during seed maturation pointed to the timing of the initiation of chlorophyll clearing as a potentially

 important factor in determining the residual chlorophyll content of mature seed.


Organisms need to adjust their cellular metabolism and growth as a consequence of changes in nutrient

 availability, developmental, and environmental signals. The capacity to monitor and respond to soluble

 carbohydrate levels is an important adaptive mechanism, and hexokinase, the key enzyme that

 catalyzes the first step in the glycolytic pathway (phosphorylation of hexose), has been implicated as a

 glucose sensor in organisms as diverse as yeasts (Entain and Fröhlich, 1984; Rose et al., 1991) and

 mammals (Efrat et al., 1994; Grupe et al., 1995). Recent results are consistent with the view that

 hexokinase is also a bifunctional enzyme in plants. In addition to phosphorylating hexoses, it acts as a

 sensor of soluble carbohydrate levels which, in turn, can activate or repress gene expression (Graham

 et al., 1994; Jang and Sheen, 1994; Jang, et al., 1997). It is apparent that soluble carbohydrates affect

 the expression of genes involved in many essential processes, such as glycolysis, glyoxylate

 metabolism, nitrogen metabolism, defense mechanisms, cell cycle regulation, sucrose and starch

 metabolism, and photosynthesis (Sheen, 1994; Koch, 1996). High carbohydrate levels repress the

 expression of genes for carbohydrate production and induce genes for storage and utilization.

 Carbohydrate depletion exerts opposite effects.


Using cellular systems, several groups have independently demonstrated that genes involved in

 photosynthesis are repressed by glucose (Harter, et al., 1993; Krapp, et al., 1993; Jang and Sheen,

 1994). Glucose transport alone is not sufficient to trigger repression. Glucose phosphorylation by

 hexokinase is required. For example, the glucose analog 3-0-methylglucose, which is transported into

 cells but not phosphorylated via hexokinase, does not trigger repression. Glycolytic intermediates

 downstream of glucose, including the immediate phosphorylated product, glucose-6-phosphate, have

 no effect on photosynthetic gene repression (Jang and Sheen, 1994). The glucose analog 2

-deoxyglucose, which is phosphorylated by hexokinase, but is not metabolized in the glycolytic pathway,

 triggers a strong repression. Further, a hexokinase-specific inhibitor is able to reduce the glucose

 repression of a maize photosynthesis-related gene (Jang and Sheen, 1994). Taken together, these

 results indicate that phosphorylation of hexose by hexokinase is the site of soluble carbohydrate

 sensing in plants (Graham et al., 1994; Jang and Sheen, 1994). However, although glucose

 phosphorylation is important, cellular glucose content does not determine the strength of the signal.

 Instead, metabolic flux through the hexokinase appears to be a critical factor. Regulatory function of a

 bifunctional hexokinase is viewed as associated with a conformational change in the enzyme that

 occurs transiently during the phosphorylation reaction.


In the present study, a yeast-derived invertase gene was introduced targeted to either the cytosol or

 apoplast and under the control of the seed-specific napin storage protein promoter. The aim was to

 increase the flux through the bifunctional hexokinase reaction by increasing the level of hexose

 substrate (glucose and/or fructose). It was anticipated that increased flux would down-regulate genes

 associated with synthesis of chloroplast differentiation, including chlorophyll a/b binding protein (Jang

 and Sheen, 1994; Sheen, 1994), and perhaps chlorophyll itself. In any event, if sufficient chlorophyll a/b

 binding sites were not available, newly synthesized chlorophyll would be degraded. By increasing

 invertase at mid-maturity, chlorophyll clearing in the maturing seed would be precociously induced and,

 because once induced the rate of clearing is more or less constant (McGregor, 1995), chlorophyll

 clearing would be completed sooner.







Brassica napus L. cv Westar was transformed with with a recombinant DNA vector consisting of

 pHS732, which is a pBIN19  derived vector (Bevan, 1984) that contains a 35S-35Spro-GUS-NPTII-nos

 selection cassette (Kay et al., 1987) and the uid A (GUS) gene (Jefferson, 1987) of E. coli fused to the

 neomycin phosphotransferase II gene. The trait gene, described in von Schaewen et al. (1990),

 consisted of the coding region of the mature protein of the Saccharomyces cerevisiae suc2 gene

 (Taussig and Carlson, 1983). Gene expresion was driven by the napin promoter from the napin gene

 BngNAP1 (Baszczynski et al., 1990) isolated from B. napus cv Westar. Transformed material was

 selected by GUS assay and for single copy insertions by Southern analysis. Invertase activity was

 determined on developing seed tissue. Homozygous seed was selected by GUS assay of seed.


Brassica napus cv Westar was also transformed with a recombinant DNA vector consisting of pRD400,

 which is a pBIN19  derived vector (Bevan, 1984) that contains the nos-wild type nptII-nos selection

 cassette (Datla et al., 1992). The trait gene, described in von Schaewen et al. (1990), consisted of an

 N-terminal sequence of the potato proteinase inhibitor II gene (Keil et al., 1986) fused in front of the

 coding region of the mature protein of the Saccharomyces cerevisiae suc2 gene (Taussig and Carlson,

 1983). Gene expresion was driven by the napin promoter from the napin gene BngNAP1 (Baszczynski

 et al., 1990) isolated from B. napus cv Westar. The proteinase inhibitor II sequence targets the yeast

 invertase to the cell wall and is cleaved during targeting. Transformed material was selected by

 kanamycin assay and for single copy insertions by Southern analysis. As many single copy

 independent transformations as possible were grown out, single plants selfed, and screened for

 homozygous transformed and homozygous non-transformed by kanamycin assay of either maturing

 seed or cotyledons of developing seedlings.


Sampling and analysis


Seed samples were collected during the filling phase of seed development to determine soluble

 carbohydrate, starch, soluble and insoluble acid invertase, and chlorophyll content. Flowers were

 tagged upon opening, brush pollinated and inflorescences bagged. Developing seed were collected

 between 27 and 45 days after pollination (DPA), frozen in liquid nitrogen, and stored at -80­%C.


Glucose, fructose and sucrose were determined enzymatically according to Stitt et al. (1989) and

 expressed in mol × g ­fresh matter-1. Starch content was determined using the pellet remaining after

 ethanol extraction according to Stitt et al. (1978) and expressed as g glucose equivalent  seed part

-1. Soluble and insoluble invertase were assayed essentially according the method of (von Schaewen et

 al., 1990). Chlorophyll content was determined using dimethylformamide (DMF) as the extraction

 solvent (Morgan and Porath, 1980; Morgan, 1982) and expressed in parts per million (ppm).





Transformation of the yeast invertase was initially targeted to the cytosol. Cytosolic expression of

 invertase was originally chosen over apoplastic (or vacuolar expression) because studies with tobacco

 had shown that plants were more sensitive to invertase expression in the cytosol, the cytosolic

 invertase activity was highly expressed, and expressed earlier in leaf development (Sonnewald et al.,

 1991). Subsequently, Frommer and Sonnewald (1995) noted that in developing potato tubers

 expression of invertase in the cytosol led to reduced starch accumulation and yield while expression of

 invertase in the apoplast led to improved tuber growth, with unaltered or only slightly reduced starch

 content. Accordingly, the yeast invertase was also targeted to the apoplast.


In total 45 primary transformants (accessions) were produced expressing a single copy of the yeast

 invertase gene targeted to the cytosol and restricted to cells in maturing B. napus cv Westar seeds with

 the seed-specific storage protein napin promoter. Selfed (R1 seed) were obtained from 44 of these

 primary transformants. Up to four R1 seeds of each independent transformation were grown out, seed

 selfed, collected 30 days post anthesis (DPA), and the immature seed analyzed for soluble and

 insoluble acid invertase activity. Homozygous lines were identified for 16 accessions. Soluble and

 insoluble acid invertase activities for 30 DPA seed of the wild type were 0.042 and 0.094 mols  min

-1 g fresh matter, respectively. For the homozygous plants, soluble and insoluble acid invertase

 ranged up to 0.527 and 0.262 mols  min g fresh matter, respectively. The highest soluble

 acid invertase represented a 12.5 fold increase over Westar. Heterozygous plants showed less

 variability in soluble acid invertase suggesting that differences would be easier to detect by screening

 homozygous plants.


In total 40 primary transformants (accessions) were produced expressing a single copy of the yeast

 invertase gene targeted to the apoplast and restricted to cells in maturing B. napus cv Westar seeds

 with the seed-specific napin storage protein promoter. Selfed (R1 seed) were obtained from 35 of these

 primary transformants.


From the earliest produced transformants both homozygous transformed and homozygous

 nontransformed seed were identified for three lines, 1781, 1785 and 2077, and, based on the relatively

 high invertase expression, these lines were selected for further study. Seed was grown out and 30 DPA

 maturing seed collected for analysis of soluble and insoluble acid invertase activity, soluble sugars

 (glucose, fructose and sucrose) and starch. Total acid invertase for individual plants ranged up to 1.548

 mols  min g fresh matter. It was noted that when insoluble acid invertase was elevated in

 the transformants soluble acid invertase was also elevated indicating that not all of the yeast invertase

 may have reached the apoplast and been bound to the cell wall. The highest total acid invertase

 represented an 11.3-fold increase over the mean for Westar and a 10.8-fold increase over the mean

 value for the nontransformed plants of the same line.


Carbohydrate analysis revealed increases in glucose and fructose and declines in sucrose for

 homozygous transformed versus non-transformed plants. The increase in glucose content was

 approximately 2-fold. Starch data showed no consistent pattern.


Seed of two homozygous lines, 1781 and 1785, were gorwn in a growth chamber at 18/15°­C day/night

 and 18/6 h light/dark regime, respectively.  And sampled at 3 day intervals between 27­and 48­days

 post anthesis (DPA). Soluble acid invertase activity was comparable for homozygous transformed and

 nontransformed plants of both 1781 (Fig. 1) and 1785 (Fig. 2) over the three week filling period.

 Insoluble acid invertase was higher in the homozygous transformed plants compared to the

 homozygous nontransformed plants for both lines (Figs. 1, 2). For both lines, chlorophyll content of

 homozygous transformed and nontransformed plants was comparable both in the peak chlorophyll

 content accumulated and in the rate and timing of its decline (Fig. 3, 4). The data indicate that

 overexpression of the yeast invertase or, at least, the level of overexpression achieved with these lines

 was not sufficient to impact on chloroplast development and the chlorophyll clearing process.



Figure 1.  Soluble and insoluble invertase activity of maturing seeds from Brassica napus cv Westar

 line 1781 homozygous non-transformed (hh) and homozygous transformed (HH) for yeast invertase

 under the control of the seed-specific napin storage protein promoter and targeted to the apoplast.




Figure 2.  Soluble and insoluble invertase activity of maturing seeds from Brassica napus cv Westar

 line 1785 homozygous non-transformed (hh) and homozygous transformed (HH) for yeast invertase

 under the control of the seed-specific napin storage protein promoter and targeted to the apoplast.




Figure 3.  Chlorophyll content of maturing seeds from Brassica napus cv Westar line 1781 homozygous

 non-transformed (hh) and homozygous transformed (HH) for yeast invertase under the control of the

 seed-specific napin storage protein promoter and targeted to the apoplast.





Figure 4.  Chlorophyll content of maturing seeds from Brassica napus cv Westar line 1785 homozygous

 non-transformed (hh) and homozygous transformed (HH) for yeast invertase under the control of the

 seed-specific napin storage protein promoter and targeted to the apoplast.



Interestingly, insoluble acid invertase activity did not decline over the filling period (Figs. 1, 2). In

 addition, it was observed that many cytosolic and apoplastic transformants germinated poorly. For

 example, of 379 R1 seeds from 27 cytosolic transformants planted to select for homozygosity, 70 failed

 to germinate, and of 518 R1 seeds from 30 apoplastic transformants planted to select for homozygosity,

 290 failed to germinate. It is possible that high invertase expresssion, particularly in the apoplast,

 impeded germination. Recently, Weber and coworkers (Weber et al., 1998) reported on attempts to

 change the sugar status in developing seed of narbon bean (Vicia narbonensis) by overexpressing a

 yeast-derived invertase gene under control of the LeguminB4 seed storage protein promoter. A signal

 sequence targeted the invertase to the apoplast in maturing embryos. In the cotyledons, sucrose was

 decreased whereas hexoses strongly accumulated, similar to the results for apoplastic expression in

 Westar. Transgenic seeds were found to germinate so poorly that Weber and coworkers were

 constrained to analyzing the segregating population of single seeds (R1). It was not possible to

 generate homozygous transgenic lines of the stronger expressors.


Soluble carbohydrates affect the expression of genes involved in many processes (Sheen, 1994; Koch,

 1996). In addition to the synthesis of chloroplast components, germination has been reported to be

 influenced by the effect of soluble carbohydrates on gene expression (Zhou et al., 1998).


Seed of a glucose-insensitive mutant identified in Arabidopsis (gin1) was recently shown to germinate

 faster (Zhou et al., 1998). Insensitivity to glucose repression of cotyledon and shoot development was

 phenocopied by ethylene precursor treatment of wild-type plants or by constitutive ethylene

 biosynthesis and constitutive ethylene signalling mutants, while an ethylene insensitive mutant

 exhibited glucose hypersensitivity. GIN1 was postulated to balance the control of plant development in

 response to metabolic and hormonal stimuli that act antagonistically. It was postulated that

 phosphorylation of glucose via hexokinase would lead to the accumulation of GIN1 which, in turn,

 would block ethylene promotion of germination (Zhou et al., 1998).


As with the overexpression of a yeast-derived invertase gene in developing seed of narbon bean

 (Weber et al., 1998), in the present study, overepression of a yeast-derived invertase in Westar may

 result in increased flux through a carbohydrate-sensing hexokinase leading to reduced germination.


Recently Trethewey and coworkers (Trethewey et al., 1998) introduced a bacterial glucokinase from

 Zymomonas mobiles into an transgenic line of potato overexpressing a yeast-derived invertase. They

 had previously noted that specific expression of a yeast invertase in the cytosol of tubers led to a

 reduction in sucrose content, a reduction in starch, and an accumulation of glucose (Sonnewald et al.,

 1997). Transgenic lines were obtained with up to threefold more glucokinase activity than in the parent

 invertase line. There was a further dramatic reduction in starch content, down to 35% of wild-type

 levels and no accumulation of glucose. Biochemical analysis of growing tuber tissue revealed large

 increases in the metabolic intermediates of glycolysis, organic acids and amino acids, two- to threefold

 increases in the maximum catalytic activities of key enzymes in the respiratory pathways, and three- to

 fivefold increases in carbon dioxide production. These changes occurred in the lines expressing

 invertase, and were accentuated following introduction of the second transgene, glucokinase.

 Trethewey and coworkers concluded that the expression of invertase in the cytosol of potato tuber cells

 leads to an increased flux through the glycolytic pathway at the expense of starch synthesis and that

 heterologous overexpression of glucokinase enhances this change in partitioning.


In a further study Trethewey and coworkers (Trethewey et al., 1999) evaluated whether the localization

 of sucrose cleavage had an impact on the glycolytic induction. Three additional transgenic potato lines

 were used, one expressing ADP-glucose pyrophosphorylase in the antisense configuration, and two

 double transgenic lines overexpressing a yeast-derived invertase targeted to either the cytosol or

 apoplast specifically in tubers of the ADP-glucose pyrophosphorylase antisense line. It was found that

 induction of the glycolitic enzymes only occured when the invertase was targeted to the cytosol, and

 that the extent of this induction was comparable when invertase was overexpressed in the cytosol of in

 the wild type (Sonnewald et al., 1997) or antisense ADP-glucose pyrophosphorylase backgrounds.

 These results contrasted those of Herbers and coworkers (Herbers et al., 1996) who showed that

 activation of plant defence mechanisms and repression of expression of photosynthetic genes occurred

 when a yeast invertase was localized in the apoplast of tobacco leaves and not when it was targeted to

 the cytosol. Trethewey and coworkers (Trethewey et al., 1999) conclude that the signal regulating

 glycolysis is directly linked to cytosolic sucrose hydrolysis and hypothesised that signalling may be

 associated with low cytosolic sucrose rather than flux through the hexokinase reaction per se.


Taken together, these studies would seem to indicate that if invertase is overexpressed in the cytosol,

 storage capacity may be limited as partitioning is directed towards respiration (glycolysis). Repression

 of photosynthetic gene expression is unlikely to occur because the cytosolic hexokinase in not

 bifunctional. On the other hand, if invertase is overexpressed in the apoplast, repression of

 photosynthetic genes may enhancing chlorophyll turnover but elevated invertase activity in the

 developing seed must dissipate before seed maturity in order not to interfere with germination.



Technical assistance of D. Puttick, W. Friesen, G. Nowak, S. Campbell, D. Capcara and R. Wood is

 greatfully appreciated. Financial assistance was received from the Canola Council of Canada and

 Agriculture and Agri-Food Canada Matching Investment Initiative.



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