Polymerase Chain Reaction and Nucleic Acid Hybridization

Supplementary Material

Supplementary Methods

Polymerase chain reaction and nucleic acid hybridization

Polymerase chain reaction was used in amplifying the nptII and AnnBj1 fragments for preliminary assessment of the transgenic plants. Genomic DNA (10 μg) was digested completely with chosen restriction enzyme(s), restriction fragments separated by agarose gel electrophoresis and transferred on to a Hybond-N+nylon membrane (Amersham Biosciences, Little Chalfont, UK), and UV cross linked. The coding region of AnnBj1 (~1 kb) and nptII fragments (~0.7 kb) were gel-extracted using Perfectprep Gel-cleanup kit (Eppendorf, Germany), labeled with [α-32P] dCTP using the ‘Prime-a-gene labeling kit’ (Promega, Madison, USA). The Southern and Northern hybridizations were performed following standardmolecular biology protocols (Sambrook et al.1989).

Estimation of relative water content (RWC)

Relative water content of WT and transgenic seedlings after different stress treatments were determined as described below:

RWC = (Fresh weight – Dry weight)/ (Turgid weight – Dry weight) ×100

Fresh weight was determined before the initiation of treatment. The dry weight was obtained after drying the seedlings for 48 h at 72°C. Turgid weight was determined from the seedlings after incubation with double distilled water for 72 h.

Supplementary Table

Supplementary Table S1 Segregation analysis in progeny of T0 transgenic cotton lines as assessed by kanamycin sensitivity test

a Kanamycin (75mg l-1) was supplemented in half-MS liquid medium for seed germination.

bχ2-values obtained by segregation analysis on the data of germinated seedlings on kanamycin indicate that the T1 progeny followed Mendelian 3:1 ratio at P0.05.

Supplementary Table S2Average root parameters in stress treatments of WT and T2 transgenic cotton seedlings with and without stress. The different treatments include mock (H2O), 100 mM mannitol, 10% PEG and 10 mM H2O2. Values representmean±SE.

a,b,cMean ±SEvalues represented by different superscripts differ significantly at P0.05.
Supplementary Table S3 Estimation of peroxidase activities and H2O2 content from the leaf discs of mature plants. The different treatments include mock (water), 100 mM mannitol, 10% PEG and 10 mMH2O2. The values represent mean ± SE. Asterisk represents significantly different values at P<0.05.

Legends to supplementary figures

Figure S1A Amino acid sequence alignment of annexin in Brassica junceashowing 71% and 91% identity with Gossypium hirsutum and Arabidopsis thaliana annexin proteinsrespectively. Amino acids highlighted with black color represent the identity whereas grey color shade represents the conserved and semi-conserved substitutions in the amino acids.

Figure S1B The phylogram showing the phylogenetic relationships of the deduced amino acid sequences of annexin proteins from plants. The phylogram was constructed using Drawgram 3.66 program and the alignment generated from clustalWwas used as input. The annexin sequences used in this analysis are:Arabidopsis thaliana (AF083913); Brassica juncea (AY356355); Zea mays (X98244); Fragaria x ananassa (AF188832); Medicago truncatula (Y15036); Nicotiana tabacum (AF113545); Gossypium hirsutum (U89609); Lavatera thuringiaca (AF006197); Capsicum annuum32 (X93308); Solanum tuberosum (AJ401032) and Lycopersicum esculentum (AF079232).

Figure S1C Alignment of plant amino acid sequences.

The following sequence data were obtained from GenBank/NCBI/EMBL RefSeq data libraries with the accession numbers: Human annexin- 5 Anx5 (U05760) and HRP (Armoracia rusticana horseradish peroxidase, CAA00083). Amino acid sequence alignment was performed using ClustalW and edited in JalView ( Clamp et al. 2004). The sequences highlighted are as follows: red, shows the heme binding motif of peroxidase from Armorecia rusticana (HRP/140-171) with the N- terminus of annexins with identical residues highlighted and the conserved His residue marked (asterisks); blue, the S3 clusters (Hofmann et al. 2003) putatively involved in redox reactions; green, salt bridges involved in channel function of animal annexins; pink, endonexin (type II Ca2+ binding) sequences. The putative annexin repeats (Repeat 1-4) were shown as a red colored underscoring beneath the sequences (SMART analysis). The presence of His40 residue is identified in the sequence comparison analysis of AnnBj1 with other annexins and horse radish peroxidase. The annexin sequences are ANNBj1/1-317; ANX A5/1-320; ANNGh1/1-316; ANN At1/1-317, ANN Ca24/1-314; ANN Zm33/1-314.

Figure S2 Leaf disc senescence assay, (A)and (B) Leaf disc assay of WT and transgenic plants at mature stage on different concentrations of NaCl and H2O2. There is visible deterioration of chlorophyll pigment in WT leaf discs and this effect is comparatively minimal in transgenic plants.Leaf discs on (C) Mannitol (100 mM- 400 mM) and (D) Polyethylene glycol (10%-40%). Damage to the leaf discs was more in WT plants when compared transgenic plants.

Figure S3 The chlorophyll content (µg mg-1 FW) in the leaf-discs after 72 h of different treatments: (A) Mannitol (100mM-300mM); (B) PEG (10%-40%) and (C) H2O2 (10mM-40mM). In each treatment, values of transgenic plants are significantly different from WT (P0.05).

Figure S4 TBARS (µmol g-1FW) in leaf-discs after 72 h of different treatments:(A) Mannitol (B) PEG and (C) H2O2. Values of transgenic plants are significantly different from WT plants (P0.05).

Figure S5 (E) Fiber cross sections (30 DPA) of WT and transgenic plants (4-3, 5-7 and 11-1). Flat ribbon shaped immature cells are prevalent with thin secondary walls, cellulose deposition and lesser theta in WT. In 5-7, showing mature, circular fibers with thick secondary walls and higher theta are prevalent (Bar-5µm).

Supplementary Figure S1

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Figure S3

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Figure S5

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