Volume 5, Issue 2, Pages 430-441 (March 2012) Overexpression of a Potato Sucrose Synthase Gene in Cotton Accelerates Leaf Expansion, Reduces Seed Abortion, and Enhances Fiber Production  Shou-Min Xu, Elizabeth Brill, Danny J. Llewellyn, Robert T. Furbank, Yong-Ling Ruan  Molecular Plant  Volume 5, Issue 2, Pages 430-441 (March 2012) DOI: 10.1093/mp/ssr090 Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 1 Flow Chart of Transgenic Analyses (A) and Representative Results of Southern Blot Hybridization to Determine Transgene Copy Number at T0 Generation (B) and PCR Screening for Transgene Segregation at T1 Generations from line 100–1 (C). For Southern blot analysis, a 292-bp 3'UTR fragment of the potato Sus cDNA was used as a probe to hybridized with genomic DNA digested with KpnI and ScaI. PCR screening was conducted by using a pair of primers, generating a 0.65-bp fragment spanning the region of S7 promoter and potato Sus cDNA. Numbers in (B) and (C) represent transgenic events at T0 generation and 18 T1 segregants from transgenic line 100–1, respectively. Note, among the 18 T1 segregants screened in (C), 14 were PCR-positive and four were PCR-negative, resulting in a segregation ratio of 3.5:1.0, consistent with the single-copy nature of the line 100–1. WT, wild-type. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 2 Sus and Acid Invertase Activities (A) and Seed Weight (B) Measured from 0-d Seed of the Transgenic and Null Segregants from Seven Selected Lines at T1 Generation. Insert in (A) is a Western blot showing that Sus protein extracted from 0-d seed appeared more abundant in transgenic plant 267–9 than its null segregant 267–16n. The blot was probed by a polyclonal antibody against a rice Sus with each lane loaded with 5 μg of protein. Numbers on the x-axis indicate one to two T1 transgenic individuals and a null segregant (n) derived from each of seven transgenic lines (267, 100–1, 65, 292, 152, 168, and 168–1). Note, a co-suppression line T071 was a dwarf and sterile and has been kept through cuttings. Each value is the mean ± SE of three biological replicates. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 3 Quantitative Relationship between Changes in Sus Activity and 0-d Seed Weight. Data were derived from Figure 2. Circle indicates where the wild-type values lie. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 4 Segregation Analyses of T1 Segregants for Impact on Fiber Elongation by Overexpression of Sus. (A) Northern blot analyses of potato–Sus transgene mRNA level in T1 segregants of transgenic lines 100–1 and 267. Note, the transgene transcript was detected only in the transgenic progeny of 100–1–7 and –9 and 267–1, but not in their respective null segregants (labeled with *) of 100–1–12 and –19 and 267–16. Arrows indicate transgenic lines used for enzyme assay (D) and fiber length measurement (E). (B) The blot of (A) was stripped and re-hybridized with endogenous GhSusA cDNA probe. Note, endogenous GhSusA transcript was detected in all plants, albeit with variable levels. (C) Near equal loading of the RNA samples was indicated by the ethidium bromide-stained rRNA bands in each lane. (D) Sus activity in 20-d fiber harvested from high and low expressers of the potato Sus transgene, 267–1 and 100–1–9 and their nulls. (E) Fiber length at 20 DAA from high and low expressers of the potato Sus transgene, 267–1 and 100–1–9 and their nulls. Each value in (D) and (E) represents mean ± SE of four biological replicates. Different letters in (D) and (E) indicate significantly different at P ≤ 0.05 according to randomization one-way ANOVA test. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 5 Vegetative Growth Phenotype of Sus-Overexpressing T1 Transgenic Segregants (267–1 and 100–1–9) about 1 Month after Germination. Note accelerated vegetative growth of the transgenic plants in comparison with their respective nulls. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 6 Sus and Invertase Activities and Sugar Levels in Developing Cotton Leaves of Homozygous T2 Progeny Lines Overexpressing the Potato Sus Gene. (A) Growth phenotype in a representative Sus-overexpression line 267–1 and its null, showing enhanced vegetative growth in the transgenic line prior to flowering. Red circles indicate shoot apices for harvesting emerging leaves for enzyme (C) and sugar (D) assays. (B) Sus and invertase activities in almost fully expanded leaves at ∼80% of their final area from multiple transgenic lines and their respective nulls and wild-type plants. n1, 2, and 3 were nulls from lines 267, 100–1, and 292, respectively. (C) Sus and invertase activities in emerging leaves at ∼10% of final area between transgenic line 267–1 and its null. (D) Sugar levels in emerging leaves at ∼10% of final area between transgenic line 267–1 and its null. Each value in (B), (C), and (D) represents mean ± SE of four biological replicates. Asterisks indicate significant difference (Student’s t-test, * P ≤ 0.05; ** P ≤ 0.01) between transgenic plants and their respective nulls. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 7 Sus and Invertase Activities in 20-d Cotton Fibers and Fiber Length, Mature Seed Number, and Fiber Yield of Homozygous T2 Progeny Lines Overexpressing the Potato Sus Gene. (A) Sus and invertase activities in 20-d fibers from multiple transgenic lines and their respective nulls and wild-type plants. n1, 2, and 3 were nulls from lines 267, 100–1, and 292, respectively. (B–D) 20-d fiber length, mature seed number per boll, and fiber yield per boll, respectively, from multiple transgenic lines and their respective nulls and wild-type plants. n1, 2, and 3 were nulls from line 267, 100–1, and 292, respectively. Each value represents mean ± SE of four biological replicates. Asterisks indicate significant difference (Student’s t-test, * P ≤ 0.05; ** P ≤ 0.01) between transgenic plants and their respective nulls. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions

Figure 8 Sus and Invertase Activities (A) and Sugar Levels (B) in 10-d Cotton Fibers of Sus Overexpressing Homozygous T2 Progeny Line 267–1 in Comparison with Null Plants. Asterisks indicate significant difference (Student’s t-test, * P ≤ 0.05; ** P ≤ 0.01) between transgenic plants and their respective nulls. Molecular Plant 2012 5, 430-441DOI: (10.1093/mp/ssr090) Copyright © 2012 The Authors. All rights reserved. Terms and Conditions