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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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CWINV4 is highly expressed in nectaries. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression profiles of AtCWINV4 (A) and an orthologue from Brassica rapa (B; BrCWINV4, accession number GQ146458). The tissues examined in (A) included: (1) petal; (2) sepal; (3) rosette leaf; (4) stamen; (5) pistil; (6) root; (7) internode shoot; (8) silique; (9) mature median nectaries (Stage 14–15); (10) immature lateral nectaries (Stage 11–12); and, (11) mature lateral nectaries (Stage 14–15). (B) Tissues included: (1) petal; (2) sepal; (3) leaf; (4) stamen; (5) pistil; (6) root; (7) stem; (8) mature median nectaries; and (9) mature lateral nectaries. GAPDH (At3g04120) and a B. rapa ubiquitin gene (BrUBQ, accession number GR719937) were used as constitutively expressed controls. All B. rapa floral tissues examined were from the equivalent of Stage 14–15 Arabidopsis flowers. The images shown are the results obtained after 27 cycles of RT-PCR. (C) Bright field image of Stage 15 Arabidopsis flower expressing an AtCWINV4:GFP fusion, under control of the native AtCWINV4 promoter, and (D) GFP fluorescence derived from same flower. Sepals were removed prior to imaging. Re, receptacle; Pe, petal; LS, long stamen; MN, median nectary; LN, lateral nectary; scale bars, 100 μm.
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fig1: CWINV4 is highly expressed in nectaries. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression profiles of AtCWINV4 (A) and an orthologue from Brassica rapa (B; BrCWINV4, accession number GQ146458). The tissues examined in (A) included: (1) petal; (2) sepal; (3) rosette leaf; (4) stamen; (5) pistil; (6) root; (7) internode shoot; (8) silique; (9) mature median nectaries (Stage 14–15); (10) immature lateral nectaries (Stage 11–12); and, (11) mature lateral nectaries (Stage 14–15). (B) Tissues included: (1) petal; (2) sepal; (3) leaf; (4) stamen; (5) pistil; (6) root; (7) stem; (8) mature median nectaries; and (9) mature lateral nectaries. GAPDH (At3g04120) and a B. rapa ubiquitin gene (BrUBQ, accession number GR719937) were used as constitutively expressed controls. All B. rapa floral tissues examined were from the equivalent of Stage 14–15 Arabidopsis flowers. The images shown are the results obtained after 27 cycles of RT-PCR. (C) Bright field image of Stage 15 Arabidopsis flower expressing an AtCWINV4:GFP fusion, under control of the native AtCWINV4 promoter, and (D) GFP fluorescence derived from same flower. Sepals were removed prior to imaging. Re, receptacle; Pe, petal; LS, long stamen; MN, median nectary; LN, lateral nectary; scale bars, 100 μm.

Mentions: To confirm the nectary-enriched expression of AtCWINV4, reverse transcription polymerase chain reaction (RT-PCR) was performed on RNA isolated from 11 major tissues, including nectaries. Results shown in Fig. 1A demonstrate that AtCWINV4 was highly expressed in both the lateral and median nectaries of flowers at pre- and post-anthesis (lanes 9–11); it was also expressed in pistils and roots at much lower levels. Similar expression patterns were also observed for a CWINV4 orthologue from Brassica rapa (BrCWINV4, Fig. 1B).


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

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

CWINV4 is highly expressed in nectaries. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression profiles of AtCWINV4 (A) and an orthologue from Brassica rapa (B; BrCWINV4, accession number GQ146458). The tissues examined in (A) included: (1) petal; (2) sepal; (3) rosette leaf; (4) stamen; (5) pistil; (6) root; (7) internode shoot; (8) silique; (9) mature median nectaries (Stage 14–15); (10) immature lateral nectaries (Stage 11–12); and, (11) mature lateral nectaries (Stage 14–15). (B) Tissues included: (1) petal; (2) sepal; (3) leaf; (4) stamen; (5) pistil; (6) root; (7) stem; (8) mature median nectaries; and (9) mature lateral nectaries. GAPDH (At3g04120) and a B. rapa ubiquitin gene (BrUBQ, accession number GR719937) were used as constitutively expressed controls. All B. rapa floral tissues examined were from the equivalent of Stage 14–15 Arabidopsis flowers. The images shown are the results obtained after 27 cycles of RT-PCR. (C) Bright field image of Stage 15 Arabidopsis flower expressing an AtCWINV4:GFP fusion, under control of the native AtCWINV4 promoter, and (D) GFP fluorescence derived from same flower. Sepals were removed prior to imaging. Re, receptacle; Pe, petal; LS, long stamen; MN, median nectary; LN, lateral nectary; scale bars, 100 μm.
© Copyright Policy - open-access
Related In: Results  -  Collection

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fig1: CWINV4 is highly expressed in nectaries. Reverse transcription-polymerase chain reaction (RT-PCR) was used to examine the expression profiles of AtCWINV4 (A) and an orthologue from Brassica rapa (B; BrCWINV4, accession number GQ146458). The tissues examined in (A) included: (1) petal; (2) sepal; (3) rosette leaf; (4) stamen; (5) pistil; (6) root; (7) internode shoot; (8) silique; (9) mature median nectaries (Stage 14–15); (10) immature lateral nectaries (Stage 11–12); and, (11) mature lateral nectaries (Stage 14–15). (B) Tissues included: (1) petal; (2) sepal; (3) leaf; (4) stamen; (5) pistil; (6) root; (7) stem; (8) mature median nectaries; and (9) mature lateral nectaries. GAPDH (At3g04120) and a B. rapa ubiquitin gene (BrUBQ, accession number GR719937) were used as constitutively expressed controls. All B. rapa floral tissues examined were from the equivalent of Stage 14–15 Arabidopsis flowers. The images shown are the results obtained after 27 cycles of RT-PCR. (C) Bright field image of Stage 15 Arabidopsis flower expressing an AtCWINV4:GFP fusion, under control of the native AtCWINV4 promoter, and (D) GFP fluorescence derived from same flower. Sepals were removed prior to imaging. Re, receptacle; Pe, petal; LS, long stamen; MN, median nectary; LN, lateral nectary; scale bars, 100 μm.
Mentions: To confirm the nectary-enriched expression of AtCWINV4, reverse transcription polymerase chain reaction (RT-PCR) was performed on RNA isolated from 11 major tissues, including nectaries. Results shown in Fig. 1A demonstrate that AtCWINV4 was highly expressed in both the lateral and median nectaries of flowers at pre- and post-anthesis (lanes 9–11); it was also expressed in pistils and roots at much lower levels. Similar expression patterns were also observed for a CWINV4 orthologue from Brassica rapa (BrCWINV4, Fig. 1B).

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