Volume 3, Issue 6, Pages 1075-1086 (November 2010) Evidence for the Role of Transfer Cells in the Evolutionary Increase in Seed and Fiber Biomass Yield in Cotton  Pugh Deborah A. , Offler Christina E. , Talbot Mark J. , Ruan Yong-Ling   Molecular Plant  Volume 3, Issue 6, Pages 1075-1086 (November 2010) DOI: 10.1093/mp/ssq054 Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 1 Morphological Characteristics of Cotton Seed Coat Transfer Cells in the Cultivated Tetraploid AD Genome Species G. hirsutum. (A) Scanning electron micrograph of a freeze-fractured cotton seed at 20 DAA, transverse view. Note the position of transfer cells (tc) at the innermost layer of the inner seed coat (isc). (B) Transverse resin section of 20-DAA cotton seed, stained with toluidine blue. Note strong staining in TCs. (C) Field emission scanning electron microscope (FESEM) image of transfer cells at 15 DAA, showing extensive flange wall ingrowths (arrows) with reticulate wall ingrowths just appearing (arrowheads). Reticulate ingrowths first appear as a band girdling the cell, which is coordinated between cells (indicated by brackets). (D) Higher-magnification view of (C) showing microfibril organization within flange wall ingrowths (arrowheads). Note microfibrils are parallel to the length of the wall ingrowth. (E) Higher-magnification view of (C) showing microfibril organization within reticulate wall ingrowths (arrowheads). Note microfibrils have no particular orientation. (F) FESEM image of transfer cells at 20 DAA showing extensive reticulate wall ingrowths (arrowheads) deposited over the flange wall ingrowth network. (G) A higher-magnification view of (E) showing the architecture of reticulate wall ingrowths. Arrows indicate growth loci. Em, embryo; f, fiber; isc, inner seed coat; osc, outer seed coat; tc, transfer cell. Bars = 1 mm (A), 200 m (B), 20 μm (C and F, bar shown in F), and 500 nm (D, E, and G, bar shown in G). Molecular Plant 2010 3, 1075-1086DOI: (10.1093/mp/ssq054) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 2 Comparison of Seed Coat Transfer Cell Wall Ingrowths of the Cultivated Tetraploid AD Genome Species G. hirsutum (A–C) with Its Two Diploid Progenitors, the A Genome, G. arboreum (D–F), and the D Genome, G. thurberi (G–I), between 10 and 20 DAA. Images are scanning electron micrographs. (A) Seed coat transfer cell (TC) of 10-DAA seed of the AD genome species. Note that no TC wall ingrowths (WIs) were visible at this stage. (B) TC of 15-DAA seed of the AD genome species. Note the extensive deposition of flange WI (arrows), with some reticulate WI just appearing (arrowheads). (C) TC of 20-DAA seed of the AD genome species, showing extensive reticulate WI (arrowheads) deposited on the flange WI network (arrows). (D) TC of 10-DAA seed of the A genome species. Note, in contrast to that in the AD genome (A), some flange WI have begun to be deposited at this stage (arrow). (E) TC of 15-DAA seed of the A genome species showing flange wall ingrowths (arrow). Note that the flanges appeared thicker but in low density as compared to that from the AD genome species at this stage (B). (F) TC of 20-DAA seed of the A genome species. Note the reticulate WIs (arrowheads) were evidently much less extensive in comparison with those in the AD genome species at this stage (C). (G) TC of 10-DAA seed from the D genome species. Note the flange WIs (arrow) were more evident than that in the A species (D). (H) TC of 15-DAA seed of the D genome species showing the lowest density of flange wall ingrowths (arrow) as compared to those in AD (B) and A (E) genome species. Notably, reticulate WIs were developed (arrowheads), which were just appearing in the transfer cells of AD (B) or absent in A (E) genome species. (I) TC of 20-DAA seed of the D genome species. Note the degree of reticulate WIs (arrowheads) appeared to remain unchanged as compared to that at the 15-DAA (H). Bars = 10 μm in (A–I). Molecular Plant 2010 3, 1075-1086DOI: (10.1093/mp/ssq054) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 3 Comparison of Biomass Yield for Combined Seed and Fiber (A), Seed Only (B), and Fiber Only (C) among the Cultivated AD Genome Species G. hirsutum and Its Two Diploid Progenitors, the A Genome, G. arboreum, and the D Genome, G. thurberi. Each value is the mean ± standard error of four replicates. Molecular Plant 2010 3, 1075-1086DOI: (10.1093/mp/ssq054) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 4 Immunolocalization of Sucrose Synthase (Sus) in Seed Coat Transfer Cells of the Tetraploid AD Genome Species G. hirsutum (A–C) and Its Two Diploid Progenitors, the A Genome, G. arboreum (D–F), and the D Genome, G. thurberi (G–I). (A) A cross-section of 10-DAA seed from the AD genome species showing TCs and surrounding regions, treated with pre-immune serum. (B) A cross-section of 10-DAA seed from the AD genome species, treated with Sus antiserum. It shows that evident fluorescent Sus signals in the transfer cells appeared mainly in the anticlanal walls (arrowheads). (C) A cross-section of 20-DAA seed from the AD genome species, treated with Sus antiserum. Note much stronger Sus signals in the transfer cells (arrowheads) as compared to that at 10 DAA (see (B)). (D) A cross-section of 10-DAA seed from the A genome species, treated with pre-immune serum. (E) A cross-section of 10-DAA seed from the A genome species, treated with Sus antiserum. Some weak Sus signals were detected in the transfer cells (arrowheads). (F) A cross-section of 20-DAA seed from the A genome species, treated with Sus antiserum. In contrast to that observed in AD genome seed (C), no or little Sus proteins were detected in the transfer cells. Evident Sus signals (arrowheads), however, were present in the epidermis of the embryo bordering the endosperm. (G) A cross-section of 10-DAA seed from the D genome species, treated with pre-immune serum. (H) A cross-section of 10-DAA seed from the D genome species, treated with Sus antiserum. Some weak Sus signals were detected in the transfer cells (arrowheads). (I) A cross-section of 20-DAA seed from the D genome species, treated with Sus antiserum. Similar to that observed in A genome seed (F), no or little Sus proteins were detected in the transfer cells. Some Sus signals (arrowheads), however, were present in the epidermis of embryo bordering the endosperm. Bar = 15 μm in (A–I). Molecular Plant 2010 3, 1075-1086DOI: (10.1093/mp/ssq054) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 5 Aniline Blue-Labeling of Callose in the Seed Coat Transfer Cells of the Tetraploid AD Genome Species G. hirsutum and Its Two Diploid Progenitors, the A Genome, G. arboreum, and the D Genome, G. thurberi, at 20 DAA. (A) A cross-section from the seed of the AD genome species, showing TCs and surrounding regions, treated with buffer only. (B) A cross-section from the seed of the AD genome species, treated with aniline blue, showing fluorescent callose (arrowheads) specifically in the transfer cells but not in adjacent inner seed coat and endosperm cells. (C) A cross-section from the seed of the A genome species, treated with aniline blue, showing no fluorescent callose in the transfer cells. (D) A cross-section from the seed of the D genome species, treated with aniline blue, showing no fluorescent callose in the transfer cells. Bar = 50 μm in (A–D). Molecular Plant 2010 3, 1075-1086DOI: (10.1093/mp/ssq054) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions

Figure 6 Immunolocalization of Callose in Seed Coat Transfer Cells of the Tetraploid AD Genome Species G. hirsutum Vibratome sections of 4 (A, B), 10 (C, D), 15 (E–G), and 20 (H, I)-DAA seed immunolabeled for callose (B, D, F, I) and stained with calcofluor white to show general cell wall structure (A, C, E, H). (A) Transfer cell precursors (tcp) from 4-DAA seed. Note absence of wall ingrowths. (B) The same section in (A) immunolabeled for callose, showing no callose in the tcp. (C) Transfer cells (tc) from 10-DAA seed. Some flange wall ingrowths started to appear (arrowheads). (D) Same section in (C) showing weak fluorescent callose signals in the transfer cells at this stage (arrowheads). (E) Transfer cells from 15-DAA seed, showing prominent wall ingrowths (arrowheads). (F) The same section in (E), showing stronger fluorescent callose signals (arrowheads) as compared to that at 10 DAA (see (D)). (G) Overlay of E and F at higher magnification. Callose labeling (white arrowheads) appears to be mostly between flange wall ingrowths (black arrowheads). (H) Transfer cells from 20-DAA seed, showing more extensive wall ingrowths (arrowheads) than at 15 DAA (E). (I) The same section in (H) showing stronger fluorescent callose signals (arrowheads) compared to that at 15 DAA (see F). Note the specific labeling of transfer cells but absence of labeling in the inner seed coat (isc) or endosperm (en). Bar = 15 μm (A–F, H, and I), bar shown in (I) and 5 μm (G). Molecular Plant 2010 3, 1075-1086DOI: (10.1093/mp/ssq054) Copyright © 2010 The Authors. All rights reserved. Terms and Conditions