Journal: Nature Communications

Article Title: Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex

doi: 10.1038/s41467-021-22661-8

Figure Lengend Snippet: a , b Aniline blue staining of mature pollen from wild-type Col-0 plants (Col-0, a ) or hemizygous pHTR10:cals3m plants ( SC-cal , b ). Arrows in ( b ) indicate callose deposition in sperm cells. Images are representative of three ( a ) or six ( b ) observations. c , d Transmission of SC-cal transgene from female gametophytes ( c ) or male gametophytes ( d ) analyzed by hygromycin resistance of T 1 plants obtained following reciprocal crosses between the SC-cal transgenic lines and wild-type Col-0 plants. Line 2 shows higher hygromycin resistance in ( c ) possibly due to the multiple insertions of the SC-cal . Numbers above the bars indicate seeds analyzed. e , f Developing seeds in Col-0 ( e ) or an SC-cal transgenic line ( f ). Images in ( e ) or ( f ) show representative of two pistils analyzed. Asterisks in ( f ) represent undeveloped seeds. Scale bars: 20 μm in ( b , c ); 1 mm in ( e , f ).

Article Snippet: A DNA fragment of the HTR10 promoter (1215 bp upstream region from the ATG of the HTR10 gene) was amplified from Col-0 genomic DNA using the primers ‘pHTR10HindIII_F’ and ‘pHTR10HindIII_R’ (Supplementary Table ), and cloned into the Hin dIII site of the pGWB501 destination vector to produce pDM252 , . pOR084, an entry clone containing mRUBY2-SYP132, was constructed as follows: (1) A pENTR/D-TOPO vector (Invitrogen) containing the mRUBY2 gene was amplified using the primers ‘FP_SpeI_F’ and ‘FP_EcoRI_R’ (Supplementary Table ); (2) the coding region of the SYP132 was amplified from a cDNA of a Col-0 seedling using the primers ‘SYP132_EcoRI_F’ and ‘SYP132_SpeI_R’ (Supplementary Table ); and (3) these two PCR products were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, MA, USA) to produce pOR084.

Techniques: Staining, Transmission Assay, Transgenic Assay

Journal: Nature Communications

Article Title: Persistent directional growth capability in Arabidopsis thaliana pollen tubes after nuclear elimination from the apex

doi: 10.1038/s41467-021-22661-8

Figure Lengend Snippet: a Schematic drawing of the single pollen-tube guidance assay. b – d Representative images of ovular-targeting pollen tubes visualized by aniline blue staining after the single pollen-tube guidance assay. Pollen tubes from wild-type Col-0 pollen (Col-0, b ). Pollen tubes from tdTomato-negative pollen ( wit1/2 SC-cal − RHT −, c ) or tdTomato-positive pollen ( wit1/2 SC-cal + RHT +, d ) from a wit1 wit2 mutant line hemizygous for genetically linked pHTR10:cals3m and pRPS5A:H2B-tdTomato (identical to the wit1/2 SC-cal RHT line shown in Fig. ). The right panels in ( d ) represent the RHT + pollen tube on the stigma; magnification of the dashed box in the upper-right is shown in the lower-right. Scale bar: 100 μm. Images are representative of 22 ( b ), 24 ( c ), or 22 ( d ) independent assays showing similar patterns. e Percentage of pollen tubes that emerged from the cut style. f Percentages of pollen tubes that entered the micropyle in the ovules. Statistical test: Bonferroni-adjusted significance test was performed after one-way ANOVA. Asterisk indicates significance ( P = 0.0438).

Article Snippet: A DNA fragment of the HTR10 promoter (1215 bp upstream region from the ATG of the HTR10 gene) was amplified from Col-0 genomic DNA using the primers ‘pHTR10HindIII_F’ and ‘pHTR10HindIII_R’ (Supplementary Table ), and cloned into the Hin dIII site of the pGWB501 destination vector to produce pDM252 , . pOR084, an entry clone containing mRUBY2-SYP132, was constructed as follows: (1) A pENTR/D-TOPO vector (Invitrogen) containing the mRUBY2 gene was amplified using the primers ‘FP_SpeI_F’ and ‘FP_EcoRI_R’ (Supplementary Table ); (2) the coding region of the SYP132 was amplified from a cDNA of a Col-0 seedling using the primers ‘SYP132_EcoRI_F’ and ‘SYP132_SpeI_R’ (Supplementary Table ); and (3) these two PCR products were assembled using the NEBuilder HiFi DNA Assembly Cloning Kit (New England Biolabs, MA, USA) to produce pOR084.

Techniques: Staining, Mutagenesis