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CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis.

Ruhlmann JM, Kram BW, Carter CJ - J. Exp. Bot. (2009)

Bottom Line: Unlike wild-type plants, cwinv4 lines did not produce nectar.Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar.Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, 1035 Kirby Drive, University of Minnesota-Duluth, Duluth, MN 55812, USA.

ABSTRACT
To date, no genes have been reported to directly affect the de novo production of floral nectar. In an effort to identify genes involved in nectar production, the Affymetrix((R)) ATH1 GeneChip was previously used to examine global gene expression profiles in Arabidopsis thaliana nectaries. One of the genes displaying highly enriched expression in nectaries was CELL WALL INVERTASE 4 (AtCWINV4, At2g36190), which encodes an enzyme that putatively catalyses the hydrolysis of sucrose into glucose and fructose. RT-PCR was used to confirm the nectary-enriched expression of AtCWINV4, as well as an orthologue from Brassica rapa. To probe biological function, two independent Arabidopsis cwinv4 T-DNA mutants were isolated. Unlike wild-type plants, cwinv4 lines did not produce nectar. While overall nectary morphology appeared to be normal, cwinv4 flowers accumulated higher than normal levels of starch in the receptacle, but not within the nectaries themselves. Conversely, wild-type, but not cwinv4, nectarial stomata stained intensely for starch. Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar. Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production. In addition, CWINV4 is probably responsible for the hexose-rich composition observed for many Brassicaceae nectars.

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Cell wall invertase activity is greatly reduced in cwinv4 flowers. Cell wall extracts were prepared from whole Stage 14–15 flowers (25 mg fresh weight) of wild-type and cwinv4 plants, which were subsequently tested for invertase activity. The results from one experiment with cwinv4-1 and cwinv4-2 plants are shown (n=3 independent preparations). Independent and repeated experiments with both cwinv4-1 and cwinv4-2 plants displayed consistent results, with varying amplitudes of total activity for both wild-type and mutant plants.
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fig3: Cell wall invertase activity is greatly reduced in cwinv4 flowers. Cell wall extracts were prepared from whole Stage 14–15 flowers (25 mg fresh weight) of wild-type and cwinv4 plants, which were subsequently tested for invertase activity. The results from one experiment with cwinv4-1 and cwinv4-2 plants are shown (n=3 independent preparations). Independent and repeated experiments with both cwinv4-1 and cwinv4-2 plants displayed consistent results, with varying amplitudes of total activity for both wild-type and mutant plants.

Mentions: Once confirmed as mutants, both cwinv4-1 and cwinv4-2 flowers were subsequently examined for extracellular (cell wall) invertase activity. Invertases catalyse the hydrolysis of the disaccharide sucrose into glucose and fructose (Berger et al., 2007; Winter and Huber, 2000b). Cell wall extracts were prepared as previously described (Wright et al., 1998) from whole flowers of cwinv4-1, cwinv4-2, and wild-type plants. Both cwinv4 lines displayed large decreases in total cell wall invertase activity (Fig. 3), whereas intracellular fractions displayed similar activities to wild-type plants (data not shown). The higher cell wall invertase activity observed in cwinv4-2 than cwinv4-1 flowers was probably due to the leaky expression of AtCWINV4 in cwinv4-2 (i.e. cwinv4-1 is a mutant, whereas cwinv4-2 is a knockdown mutant). It was also noted that cell wall invertase activity was not reduced to zero in cell wall extracts derived from flowers of either cwinv4 mutant. Whether this result is indicative of activity derived from other cell wall invertases, or represents minor contamination from intracellular invertases is unknown. However, it should be noted that whole flowers were used for these assays, not isolated nectaries (i.e. other cell wall invertases may be expressed in petals, stamen, etc).


CELL WALL INVERTASE 4 is required for nectar production in Arabidopsis.

Ruhlmann JM, Kram BW, Carter CJ - J. Exp. Bot. (2009)

Cell wall invertase activity is greatly reduced in cwinv4 flowers. Cell wall extracts were prepared from whole Stage 14–15 flowers (25 mg fresh weight) of wild-type and cwinv4 plants, which were subsequently tested for invertase activity. The results from one experiment with cwinv4-1 and cwinv4-2 plants are shown (n=3 independent preparations). Independent and repeated experiments with both cwinv4-1 and cwinv4-2 plants displayed consistent results, with varying amplitudes of total activity for both wild-type and mutant plants.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC2803206&req=5

fig3: Cell wall invertase activity is greatly reduced in cwinv4 flowers. Cell wall extracts were prepared from whole Stage 14–15 flowers (25 mg fresh weight) of wild-type and cwinv4 plants, which were subsequently tested for invertase activity. The results from one experiment with cwinv4-1 and cwinv4-2 plants are shown (n=3 independent preparations). Independent and repeated experiments with both cwinv4-1 and cwinv4-2 plants displayed consistent results, with varying amplitudes of total activity for both wild-type and mutant plants.
Mentions: Once confirmed as mutants, both cwinv4-1 and cwinv4-2 flowers were subsequently examined for extracellular (cell wall) invertase activity. Invertases catalyse the hydrolysis of the disaccharide sucrose into glucose and fructose (Berger et al., 2007; Winter and Huber, 2000b). Cell wall extracts were prepared as previously described (Wright et al., 1998) from whole flowers of cwinv4-1, cwinv4-2, and wild-type plants. Both cwinv4 lines displayed large decreases in total cell wall invertase activity (Fig. 3), whereas intracellular fractions displayed similar activities to wild-type plants (data not shown). The higher cell wall invertase activity observed in cwinv4-2 than cwinv4-1 flowers was probably due to the leaky expression of AtCWINV4 in cwinv4-2 (i.e. cwinv4-1 is a mutant, whereas cwinv4-2 is a knockdown mutant). It was also noted that cell wall invertase activity was not reduced to zero in cell wall extracts derived from flowers of either cwinv4 mutant. Whether this result is indicative of activity derived from other cell wall invertases, or represents minor contamination from intracellular invertases is unknown. However, it should be noted that whole flowers were used for these assays, not isolated nectaries (i.e. other cell wall invertases may be expressed in petals, stamen, etc).

Bottom Line: Unlike wild-type plants, cwinv4 lines did not produce nectar.Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar.Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, 1035 Kirby Drive, University of Minnesota-Duluth, Duluth, MN 55812, USA.

ABSTRACT
To date, no genes have been reported to directly affect the de novo production of floral nectar. In an effort to identify genes involved in nectar production, the Affymetrix((R)) ATH1 GeneChip was previously used to examine global gene expression profiles in Arabidopsis thaliana nectaries. One of the genes displaying highly enriched expression in nectaries was CELL WALL INVERTASE 4 (AtCWINV4, At2g36190), which encodes an enzyme that putatively catalyses the hydrolysis of sucrose into glucose and fructose. RT-PCR was used to confirm the nectary-enriched expression of AtCWINV4, as well as an orthologue from Brassica rapa. To probe biological function, two independent Arabidopsis cwinv4 T-DNA mutants were isolated. Unlike wild-type plants, cwinv4 lines did not produce nectar. While overall nectary morphology appeared to be normal, cwinv4 flowers accumulated higher than normal levels of starch in the receptacle, but not within the nectaries themselves. Conversely, wild-type, but not cwinv4, nectarial stomata stained intensely for starch. Cell wall extracts prepared from mutant flowers displayed greatly reduced invertase activity when compared with wild-type plants, and cwinv4 flowers also accumulated significantly lower levels of total soluble sugar. Cumulatively, these results implicate CWINV4 as an absolutely required factor for nectar production in the Brassicaceae, specifically by maintaining constant sink status within nectaries, thus allowing them to accumulate the sugars necessary for nectar production. In addition, CWINV4 is probably responsible for the hexose-rich composition observed for many Brassicaceae nectars.

Show MeSH