1. Volume 6, Issue 1, Pages 216-225 (January 2013)
NAD+ Accumulation during Pollen Maturation in Arabidopsis Regulating Onset of Germination 
Shin-nosuke Hashida, Hideyuki Takahashi, Kentaro Takahara, Maki Kawai-Yamada, Kazuyoshi Kitazaki, Kazuhiro Shoji, Fumiyuki Goto, Toshihiro Yoshihara, Hirofumi Uchimiya 
Molecular Plant 
Volume 6, Issue 1, Pages (January 2013)
DOI: /mp/sss071
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

2. Figure 1
Pathway Common to De Novo or Salvage NAD Biosynthesis. Schematic diagrams outlining the common pathway of de novo and salvage NAD+ biosynthesis, intersecting at NaMN synthesis. NaMN, nicotinate mononucleotide; NaAD, nicotinate adenine dinucleotide; Nam, nicotinamide; Na, nicotinate; NMNAT, nicotinate mononucleotide adenyltransferase; NADS, NAD+ synthetase; NADK-1, -2, NAD+ kinase-1, -2; NADK-3, NADH kinase-3; NIC, nicotinamidase; NaPRT, nicotinate phosphoribosyltransferase.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

3. Figure 2
Pollen with Altered Balance of Nicotinamide Nucleotides Pool.(A) NaAD:NaMN ratio.(B) NAD+:precursor (NaMN + NaAD) ratio.(C) NADP(H):precursor (NaMN + NaAD) ratio.(D) NAD(P)H:NAD(P)+ ratio.(E) NADP(H):NAD(H) ratio. Ratio was calculated based on same data as shown in Table 1 and indicated inside bar. Data are means ±SE of three independent experiments. Black and white bars refer to the results of freshly harvested dry pollen and pollen 7min after imbibitions, respectively. The asterisk indicates that the mean value is significantly different between before and after pollen imbibition.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

4. Figure 3
Dynamics of Nicotinamide Nucleotides in Response to Pollen Imbibition.(A) Changes in the levels of NAD+ and NADP+ during imbibition.(B) Changes in the levels of NaMN, NaAD, NADH, and NADPH during imbibition. Analyses were performed on imbibed pollen sampled at indicated time point and quantified. Open circle, NAD+; closed circle, NADP+; open triangle, NaMN; closed triangle, NaAD; open square, NADH; closed square, NADPH. Closed diamond symbol shown in (A) indicates change in NAD+ in the presence of NAD+-consuming reaction inhibitors (see the ‘Methods’ section). Data are mean ±SE of three independent experiments.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

5. Figure 4
Germination Retardation in the Presence of NAD+-Consuming Reaction Inhibitors.(A) Germination kinetics of pollen in the presence of NAD+-consuming reaction inhibitors. Each point is mean ±SE of three independent experiments: 300 pollen grains were scored per single experiment. Closed circle, control experiment without any inhibitors; open circle, value in the presence of mixture of three inhibitors.(B) The levels of NAD+, NADH, NADP+, and NADPH in dry pollen (imbibition, –) and 60-min-imbibed pollen (imbibition, +) in the presence (+) or absence (–) of NAD+-consuming reaction inhibitors.(C) Means the relative value shown in (B). Value of dry pollen is set to 100 each. Data are means ±SE of three independent experiments.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

6. Figure 5
Impairment of Tube Growth by Excess NAD+. Frequency distribution of pollen tube growth loaded with excess NAD+. Histogram showing the distribution of pollen tube growth 16h after incubation.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

7. Figure 6
Precocious Pollen Germination Inside Anther Deficient in NAD+ Accumulation.(A, B) A typical pollen grain extending a single tube of Col-0 (A) and nmnat/+ (B) on the stigma. Pollinated pistil was stained with aniline blue and the pollen on the stigma was photographed. Callose was contained within a nmnat pollen grain (arrowhead).(C, D) Pollen grain of Col-0 (C) and nmnat/+ (D) with protrusion frequently observed (arrowhead).(E, F) Effects of high humidity on Col-0 (E) and nmnat/+ (F). High humidity culture for 2d enhanced the growth of pollen tubes inside nmnat/+ anther. Inset indicates auto-fluorescence of pollen stuffed within the locule. Scale bar in (B)=30μm.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
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8. Figure 7
Promotion of nmnat/+ Sterility under High Humidity. (A) Siliques harvested from 8-week-old Col-0 (left) and nmnat/+ (right) plants grown under high humidity (40–60%) conditions.(B) Length of silique harvested from 8-week-old plants grown under low humidity (20–40%) and high humidity (40–60%). As control experiments, Col-0 and nmnat/+ were emasculated. Scale bar in (A)=2mm.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions

9. Figure 8
Pollen Viability of Col-0 and nmnat/+.(A) FDA and PI double-stained pollen grain. Freshly harvested Col-0 and nmnat/+ pollen was stored under dehydrated conditions and a portion of the pollen was sampled from storage after 3 and 48h, stained with a mixture of FDA (0.01%) and PI (0.1%) for 10min, and then observed under our fluorescence microscope (DMR, Leica). FDA positive (viable) and PI positive (inviable) pollen were indicated as green and red color, respectively.(B) Pollen viability after storage. Pollen viability was tested with FDA and PI double stain as described in the ‘Methods’ section. Each point represents the percentage of pollen stained FDA positive and PI negative. Data are mean ±SE of four individual experiments: 300 pollen grains were scored per single experiment. Open circle indicates the data obtained from Col-0. Closed square indicates the data obtained from nmnat/+. Light and dark gray field indicates SE ranges of Col-0 and nmnat/+, respectively. Vertical dotted lines indicate the time points crossing to 50% viability, half-life time.
Molecular Plant 2013 6, DOI: ( /mp/sss071)
Copyright © 2013 The Authors. All rights reserved. Terms and Conditions