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2004 12th AAC, 4th ICSC
Poster Papers
3. Genetics
Biotechnology For A Better World
Glyphosate Alters Floral Morphology In Glyphosate-resistant Cotton

Glyphosate alters floral morphology in glyphosate-resistant cotton

John Wilcut, Wendy Pline and Keith Edmisten

Crop Science Department, North Carolina State University; john_wilcut@ncsu.edu

Abstract

Glyphosate treatments to glyphosate-resistant (GR) cotton have been associated with poor pollination and increased boll abortion. In comparison to non-treated plants, glyphosate applied at both the 4-leaf stage postemergence (POST) and at the 8-leaf stage postemergence-directed (PDIR) inhibited the elongation of the anther column and filament, which increased the distance from the anthers to the receptive stigma tip 4.9 to 5.7 mm during the first week of flowering. The increased distance from anthers to stigma resulted in 42% less pollen deposited on stigmas of glyphosate-treated plants than non-treated plants. The presence of the GR CP4-EPSPS enzyme was quantified in reproductive and vegetative tissues using ELISA. Content of CP4-EPSPS in the stigma, anther, pre-anthesis floral bud (square), and flower petals were significantly less than in vegetative leaf tissue. Glyphosate affected male reproductive development and resulted in poor pollen deposition on the stigma, as well as production of aborted pollen with reduced viability. These responses to glyphosate provide a likely explanation for reports of increased boll abortion, and pollination and seed set problems in glyphosate-treated GR cotton.

Media summary

This research shows that reduced seed set and yield reductions occasionally seen with glyphosate treatments on glyphosate-reduced cotton, results from lower CP4-EPSPS expression in the male reproductive structures of the cotton flower.

Key Words

Herbicide-resistant crops, transgenic crops, reproductive biology, cotton, glyphosate.

Introduction

Since its commercialization in 1997, concern over reproductive tolerance of GR cotton to glyphosate has been raised. Numerous reports of increased boll abscission and pollination problems in response to glyphosate applications have occurred in cotton, occasionally leading to yield loss and a modified fruiting pattern (Jones and Snipes 1999). Glyphosate resistance has been conferred to cotton by the incorporation of a glyphosate-resistant 5-enolpyruvylshikimate-3-phosphate synthase (CP4-EPSPS) gene cloned from Agrobacterium sp. strain CP4. The expression of the CP4-EPSPS gene produces a glyphosate-resistant EPSPS enzyme, which can bypass the inhibition of native EPSPS in the presence of glyphosate, allowing sufficient production of aromatic amino acids and secondary metabolites (Nida et al. 1996). The objectives of the current study were to characterize differences in floral anatomy, pollen deposition, and pollen morphology between glyphosate-treated and non-treated GR and conventional cotton, and to quantify the level of CP4-EPSPS protein in reproductive and vegetative tissues of GR cotton.

Materials and Methods

Plant material and growth conditions

‘DP 50’, ‘DP 90’, ‘DP5415 RR’ cultivars and SureGrow cultivar ‘SG 125RR’ seeds were planted in 25.4 cm pots containing a gravel-peat combination substrate and grown in a phytotron. Plants were thinned to one per pot and were watered with nutrient mixture twice daily. Glyphosate treated plants received 1,120g ai/ha glyphosate (Roundup Ultra) at the 4-leaf stage (foliar application) and at the 8-leaf stage (post-directed application to stem) according to the Roundup Ultra supplemental label for GR cotton.

Pollen and tissue sampling

All pollen and anther samples were collected between 9:00 and 11:00 am. Brightfield images of pollen and reproductive organs were captured with a digital camera. Measurements of floral anatomy (height of the stigma, height of the staminal column, distance from the highest anther to the tip of the stigma, and anther filament length in mm) were measured on all flowers three times weekly on separate days. Stigma height and staminal column height were measured from the lowest portion of the inner flower where the petals converge with the anther-containing sheath to either the stigma tip for stigma heights, or the top of the staminal column where the style is visible. The anther-stigma distance was measured from the tip of the stigma to the top anther, and is in relation to the stigma. The anther filament length was measured as the length of highest anther filament to the staminal column.

Pollen deposition on stigma

Stigmas from flowers used for pollen viability assessment were harvested 24 hr after anthesis by cutting at the stigma base. The sheath containing anthers was removed from the stigma, and stigmas were placed into microfuge tubes containing a 1-mL solution of ethanol: chloroform: acetic acid (6:3:1). Stigmas were vortexed to remove loose pollen grains from the stigma surface. The stigma was removed from the microfuge tube, and pollen was pelleted by centrifuging for 1 min at 500 rpm and then resuspended. The number of pollen grains in a 25-μL subsample was then counted.

Quantification of CP4-EPSPS.

Mature leaves, fruiting branches, apical meristems, reproductive buds, and flowers were gathered from ‘DP 5415RR’ (glyphosate-resistant) and ‘DP 5415’ (conventional, glyphosate-sensitive) cotton plants grown at two separate field sites in North Carolina. Anthesis flowers were dissected into anthers (filament, anther, and pollen), stigma, ovary (capsule and ovules), and petal. CP4-EPSPS was extracted from 100 mg freshly ground tissue, diluted 5000 fold, and analyzed by ELISA using the Strategic Diagnostic, Inc. Trait Check™ kit which included directions (Anonymous 1999) for quantification of CP4-EPSPS in food products.

Results and Discussion

Glyphosate effects on floral morphology.

Differences in floral morphology between glyphosate-treated and non-treated GR cotton cultivars may explain potential differences in pollination. The variety-glyphosate treatments and week of flowering main effects were significant for pollen deposition, anther-stigma distance, stigma height, column height, and anther length measurements. Stigmas from glyphosate-treated plants were 1.2 to 1.4 mm longer than those from non-treated plants (Table 1). No other visual differences were evident between stigmas of glyphosate-treated and non-treated GR cultivars or conventional cultivars. Differences in the male reproductive organs of glyphosate-treated and non-treated GR cultivars however, were evident. The column to which the anther filaments are attached were 2.7 and 2.0 mm shorter in DP 5415RR glyphosate-treated GR plants than non-treated GR plants in the 1st and 2nd wk of flowering, respectively (Table 1). In the first 4 wk of flowering, the anther filaments were 0.8 to 0.9 mm shorter than those from non-treated plants (Table 1). The shorter anthers and anther columns, along with slightly elongated stigmas, resulted in a 4.9 to 5.7 mm greater distance from the top anther to the tip of the stigma in glyphosate-treated than non-treated plants in the first week of flowering (Table 2). Non-treated and conventional cotton plants had anther-stigma distances of –0.4 mm (anthers above stigma) to 2.9 mm. Close proximity of anthers and the stigma would allow adequate pollen coverage of the receptive stigma.

Table 1. Heights of anther column, stigma and anther length of conventional, GR, and glyphosate-treated GR cotton cultivars at 1-4 wk of flowering.


	Anther column height							Stigma height			Anther length
	Week of flowering
Cultivar-treatment	1		2		3		4		1-4		1-4
	^{___________________________________________________}mm^{_____________________________________}
DP50	15.3	a^a	13.9	bc	14.5	bc	14.9	bc	23.2	cd	7.4	a
DP 90	12.8	ab	15.5	a	15.5	a	14.7	c	21.3	e	7.2	a
DP 5415RR	13.2	ab	14.4	abc	14.8	abc	14.7	c	22.2	de	6.3	bc
DP 5415RR Trt.	10.5	c	12.4	d	13.9	c	14.8	c	23.4	cd	5.4	d
SG 125RR	13.8	ab	15.3	ab	15.2	ab	15.7	a	24.8	b	6.9	ab
SG 125RR Trt.	11.5	bc	13.7	cd	14.9	ab	15.5	ab	26.2	a	6.1	cd

^aMeans followed by the same letter in each column are not significantly different at alpha = 0.05 according to Fisher’s Protected LSD test.

Glyphosate effects on pollen deposition

Differences in floral morphology between glyphosate-treated and non-treated GR cotton cultivars explain differences in pollination, boll retention, and seed set. Male reproductive organs in GR cotton were highly affected by glyphosate applications. Anthers from non-treated GR cotton plants were covered with pollen grains at anthesis, whereas those from glyphosate-treated GR cotton plants either exposed no pollen, or malformed pollen (data not shown). Glyphosate-treated plants had 42.2 to 42.5% less loose pollen per stigma than non-treated GR plants (Table 2). Pollen deposition on the stigma of glyphosate-treated GR cotton was likely reduced because of the greater distance between the anthers and stigma (Table 2). There was a highly negative correlation (-0.98) between pollen deposition as measured by removal of loose pollen, with anther-stigma distances, demonstrating the association of greater anther-stigma distances with lower amounts of pollen deposition on the stigma. Pollen from glyphosate-treated GR plants was typically deposited only on the lower portion of the stigma (data not shown). Iyengar (1938) reported that pollen placed on the lower portion of the cotton stigma germinated much less readily than pollen placed at the top portion of the stigma, which may lead to an even lower number of pollen tubes and seed set in treated plants (Pline et al. 2003).

Table 2. Distance from top anther to tip of stigma and number of loose pollen grains per stigma from conventional, non-treated GR, and glyphosate-treated GR cotton varieties at 1-4 wk of flowering.


Anther to stigma distance								Loose pollen/stigma
Week of flowering
Cultivar-treatment	1		2		3		4		1-4
	^{_____________________}mm^{____________________}								^___pollen grains^____
DP50	2.0	b^a	-0.2	c	-0.4	e	0.1	c	1323	b
DP 90	-0.4	c	-1.9	d	-2.0	f	-1.4	d	1843	a
DP 5415RR	1.8	b	1.3	b	0.6	d	1.0	b	1120	bc
DP 5415RR Trt.	7.5	a	4.6	a	3.0	b	2.3	a	647	d
SG 125RR	2.9	b	1.9	b	1.8	c	2.4	a	857	cd
SG 125RR Trt.	7.8	a	4.8	a	3.6	a	2.7	a	493	d

^aMeans followed by the same letter in each column are not significantly different at alpha = 0.05 according to Fisher’s Protected LSD test.

CP4-EPSPS in tissues

Analysis of CP4-EPSPS content in various tissues of GR cotton revealed that CP4-EPSPS content in the stigma, anther, pre-anthesis floral bud (square), and flower petals were significantly less than in vegetative leaf tissue (data not shown). CP4-EPSPS content in the ovary of anthesis GR cotton blooms was similar to that in vegetative leaves. From the current work, male reproductive organs in GR cotton appear to be only partially resistant to the effects of glyphosate, presumably due to insufficient presence of CP4-EPSPS (Pline et al. 2002b, 2002c, 2003). However, affected pollen does not seem to degenerate as has been reported for chloroplasts in glyphosate-treated plants (Mollenhauer et al. 1987; Uotila et al. 1980). Membranes are intact and no unusual organelle swelling is observed, except for distended ER. Instead, pollen development appears to be slowed or arrested at different maturation stages probably due to the localized dose and timing of glyphosate during pollen development. At anthesis, pollen from glyphosate-treated plants shows a range of phenotypes closely resembling arrested pollen from reported male-sterile mutants as well as from cotton pollen analyzed at intervals from 12 d pre-anthesis to anthesis.

The effect of glyphosate on floral morphology, as well as pollen morphology, is transitory with the most pronounced effect during the first week of flowering (data not shown). The first flowers on a cotton plant likely receive the highest dose of glyphosate because they are the strongest metabolic sinks at the time of glyphosate application. Subsequently, as the plants increase in biomass, glyphosate may be diluted, or lost from the system as early floral organs abscise. Therefore, later developing squares may not receive an inhibitory dose of glyphosate and flowers may appear similar to non-treated plants.

Conclusions

Our findings demonstrate that floral morphology is altered in GR cotton plants treated with recommended rates of glyphosate, presumably because of lower reproductive CP4-EPSPS content. Anthers and the anther column are less elongated, while the stigma length increases slightly. These morphological differences result in less pollen deposition on the stigmas of glyphosate-treated GR plants. The majority of pollen from glyphosate-treated GR plants is arrested at different developmental stages resulting in immature, or aborted pollen at anthesis (Pline et al. 2002a, 2002b). The combination of reduced pollen deposition on the stigma, along with pollen development inhibition provide a plausible explanation for reports of increased boll abscission in glyphosate-treated GR cotton.

References

Anonymous (1999). Food ingredient testing soya kit user’s guide. Rev. 111799, version 2.0. Part number 3099992. Strategic Diagnostics Inc., Newark, DE 19702.

Iyengar NK (1938). Pollen-tube studies in Gossypium hirsutum. J. Genetics 37:69-107.

Jones, MA and CE Snipes (1999). Tolerance of transgenic cotton to topical application of glyphosate. J. Cotton Sci. 3:19-26.

Mollenhauer C, Smart CC and Amrhein N (1987). Glyphosate toxicity in the shoot apical meristem of the tomato plant. I. Plastid swelling is the initial ultrastructural feature following in vivo inhibition of 5-enolpyruvylshikimic acid 3-phosphate synthase. Pestic. Biochem. Physiol. 29:55-65.

Nida DL, Kolacz KH, Buehler RE, Deaton WR, Shuler WR, Armstrong TA, Taylor ML, Ebert CC, Rogan GJ, Padgette SR, and Fuchs RL (1996). Glyphosate-tolerant cotton: Genetic characterization and protein expression. J. Agric. Food Chem. 44:1960-1966.

Pihakaski S and Pihakaski K (1980). Effects of glyphosate on ultrastructure and photosynthesis of Pellia epiphylla. Ann. Bot. 46:133-141.

Pline WA, Edmisten KL, Oliver T, Wilcut JW, Wells R and Allen NS (2002a). Use of digital image analysis, viability stains, and germination assays to estimate conventional and glyphosate-resistant cotton pollen viability. Crop Sci. 42:2193-2200.

Pline WA, Viator R, Wilcut JW, Edmisten KL, Thomas J and Wells R (2002b). Reproductive abnormalities in glyphosate-resistant cotton caused by lower CP4-EPSPS levels in the male reproductive tissue. Weed Sci. 50:438-447.

Pline WA, Wilcut JW, Duke SO, Edmisten KL and Wells R (2002c). Tolerance and accumulation of shikimic acid in response to glyphosate applications in glyphosate-resistant and nonglyphosate-resistant cotton (Gossypium hirsutum L.). J. Agric. Food Chem. 50:506-512.

Pline WA, Edmisten KL, Wilcut JW, Wells R and Thomas J (2003). Glyphosate-induced reductions in pollen viability and seed set in glyphosate-resistant cotton and attempted remediation by gibberellic acid (GA3). Weed Sci. 51:19-27.

Uotila MK, Evjen and. Iversen T–H (1980). The effects of glyphosate on the development and cell ultrastructure of white mustard (Sinapis alba L.) seedlings. Weed Res. 20:153-158.

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