1. New microsome-associated HT-family proteins from Nicotiana respond to pollination and define an HT/NOD-24 protein family 
Kondo Katsuhiko , McClure Bruce  
Molecular Plant 
Volume 1, Issue 4, Pages (July 2008)
DOI: /mp/ssn018
Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Alignment of HT- family proteins: N. alata HT-B, HT-M1, M3 and M4 and Petunia inflata PiHTL-A and B. Signal sequences, core sequences, cysteine motifs, and the asparagines-aspartate–rich ND domain are shown.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
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3. Figure 2 Distinct classes of HT-M proteins.
(A) HT-M protein alignments. Signal sequence, variable region, and cysteine motif are shown. Sequences were obtained from the following species: SI N. alata ( N. ala M1, M3, M4), SI N. bonariensis (N. bon M1, M2, M3, M4), SI N. forgetiana (N. for M1, M2, M4), SI N. langsdorfii 28B (N. lang28B M1, M3, M4), SC N. longiflora 4C15 (N. long4C15 M1, M2, M3), SC N. plumbaginifolia ( N. plum M2), and SC N. tabacum (N. tab M5, M6).
(B) Phylogenetic tree of pistil-specific HT-family proteins. The tree was generated from a Clustal W alignment using TreeView. Scale bar corresponds to 0.1 amino acid substitutions per residue.
(C) Expression of HT-M and HT-B in pistils (i.e., stigma plus style). Primers specific for HT-M1∼4 or HT-B were used to perform PT-PCR on total pistil RNA.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
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4. Figure 3 Pistil-specific HT-M gene expression.
RT-PCR analyses were performed using total RNA from the organs and developmental stages of N. alata SA2SA2 as shown. Developing pistils and anthers were collected from buds of various sizes, as shown. For reference, anthers from 0.5 cm to ∼1 cm buds are in the tetrad stage, and a fully mature flower is ∼5.5 cm.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

5. Figure 4 HT-M RNAi gene construct.
Top, schematic of the HT-M RNAi gene construct. Bottom, partial alignment of the HT-M1, M3, and M4 gene sequences is shown. Asterisks, identical bases. A translation is shown for reference to Figures 1 and 2; the signal sequence is in lowercase. The 205 bp region under the gray box was used in the RNAi construct.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
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6. Figure 5
HT-M gene silencing and pollination phenotypes of HT-M RNAi hybrids.
(A) Suppression of HT-M gene expression. The RNAi construct was transformed into N. plumbaginifolia and crossed to SI N. alata S105S105. Hybrid progeny from six independent transformants are shown. HT-M and HT-B gene expression was analyzed by RT-PCR using total pistil RNA. Untransformed SI N. alata S105S105N. plumbaginifolia, and a control hybrid are shown (left).
(B) Protein blot analysis and pollination phenotypes. Extracts were prepared from controls and six HT-M RNAi (N. plumbaginifolia×SI N. alata S105S105) hybrids. Anti-vPPase antibody was used as loading control. Pollination phenotypes were tested with N. alata S105-and SC10-pollen, and compatibility was assessed by examining style squashes stained with decolorized aniline blue. Data are presented as the number of compatible pollinations over total pollinations attempted.
(C) Sample style squashes. Shown are typical compatible and incompatible (SC10- and S105-pollen, respectively) pollinations on an untransformed control and two HT-M suppressed hybrids (A3-2 and A14-1). Few pollen tubes (pt) reach the base of the style in incompatible pollinations, while numerous pollen tubes are present in compatible pollinations. Epidermal tissue (ep) is also visible. Pollinated styles were harvested 48 h after pollination, and pollen tubes were stained with decolorized aniline blue.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
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7. Figure 6 HT-family proteins are present in a microsomal pellet.
Emasculated (E), incompatibly pollinated (SA2), and compatibly pollinated (S105) N. alata SA2SA2 pistils were harvested 48 h after pollination. Homogenized samples were centrifuged at 10,000 ×g producing a supernatant (S10). The S10 was subjected to ultracentrifugation at 156,000 ×g producing supernatant (S156) and microsomal pellet (P156) fractions. Protein samples were immunoblotted and probed with both anti-HT-M and anti-HT-B. Anti-vPPase antibody was used as a control of microsomal protein. A Coomassie Blue stained gel is shown as a loading control. Gel loading. HT-M blot: S10, S156 – 30 μg/lane; P156 – 20 μg. HT-B and vPPase blots: S10, S156 – 20 μg/lane; P156 – 10 μg. Coomassie stain: S10, S156 – 12 μg/lane; P156 – 6 μg.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
Copyright © 2008 The Authors. All rights reserved. Terms and Conditions

8. Figure 7 Conserved features of HT-family proteins.
Conserved signal sequences, C-terminal sequences, and cysteine motifs are shown. To produce a representative alignment, one sequence from each of the three groups of pistil-specific HT-family proteins is shown along with sequences from two GRPs (Arabidopsis At2g05540 and TLRP) and soybean nodulin-24. The signal sequence cleavage site is indicated by an arrow. Cysteine motifs are boxed in the C-terminal sequences.
Molecular Plant 2008 1, DOI: ( /mp/ssn018)
Copyright © 2008 The Authors. All rights reserved. Terms and Conditions