Figure 1.
GUS and GFP reporter gene analyses of NDL1 expression around the vegetative and reproductive SAMs in Arabidopsis seedlings.
(A) NDL1-GFP localization in three-day-old etiolated seedlings. GFP fluorescence is detectable in the vicinity of the SAM. (B) Panel A with an overlay of the DIC image. (C) and (D) GUS histochemical staining in eight-day-old etiolated seedlings (C) and in ten-day-old seedlings grown in the light (D). GUS staining is not detectable in the SAM. (E) GUS histochemical staining in a longitudinal section of the vegetative SAM in etiolated seedlings. (F) Enlarged view of the SAM from panel E. (G) GUS staining in a mature stamen. (H) Papillar cells showing GUS staining upon pollen germination (the red double-ended arrow points to a germinating pollen tube). Scale bars = 50 µm, Red arrows in panels A, C, D and E indicate cell zones at the periphery of the SAM.
Figure 2.
Vegetative growth phenotypes of NDL1 over-expression lines.
Arabidopsis plants over-expressing NDL1 under the control of the 35 S promoter were used for phenotypic analyses. (A) Inflorescence stems show fasciation. (B) Nascent rosettes emerging from the axils of senescing leaves (red arrows). (C) Shoots of mature plants contain additional rosettes and cauline paraclades (red arrows). (D) Vegetative growth of a Col-0 wild-type control plant. (E) Quantification of rosette and cauline paraclades in Col-0 wild-type and 35 S-NDL1 over-expression lines. Fifteen to twenty independent plants were analyzed for each genotype, and error bars represent SE. Student's t test results are based on differences between wild type and the indicated genotype, and asterisks indicate that P<0.05. (F) “Shoot upon shoot” phenotype of a Col-0 wild-type plant and an agb1-2 mutant over-expressing NDL1 under the control of the endogenous NDL1 promoter. Red arrows point to the origins of the nascent shoots.
Figure 3.
Reproductive growth phenotypes of NDL1 over-expression lines.
Twelve week old Arabidopsis plants over-expressing CFP and MYC-NDL1 under the control of the 35 S promoter were used for phenotypic analyses. (A) Ectopic over-expression of NDL1 causes a silique to emerge from another silique, which contains an open carpel bearing ovules. (B) Open siliques emerging from a single pedicel. (C) Shoots of agb1-2 mutant plants contain abnormal terminal flowers. Red arrows mark open ovules, and black arrows mark pistils emerging from a silique.
Figure 4.
Reduced expression of the NDL genes affects vegetative organ development in Arabidopsis.
Vegetative growth of ndlM mutant plants with reduced expression of all three NDL genes. (A) Altered phyllotactic pattern of ndlM mutants and Col-0 wild-type plants (inset) during early stages of development. ndlM mutants, but not Col-0 plants, show asymmetrical leaf emergence (the red arrow points to the missing partner of the leaf pair). (B) Some ndlM mutants form an Arabidopsis tricot mutant-like structure (red arrow). (C) Leaf phenotype of ndlM mutants. Some of the early rosette/vegetative leaves show defects in leaf shape and size. The arrow points to the notch of a heart-shaped leaf with an enlarged lamina. (D) More than 80% of ndlM mutant plants displayed twinning or the formation of multiple rosettes. The arrow points to the center of a twinned rosette. (E) ndlM mutant primary shoots emerging from a twinned rosette. The red arrows point to rosette paraclades.
Figure 5.
Reduced expression of NDL genes affects reproductive organ development in Arabidopsis.
Phenotypic analyses of reproductive organs from twelve-week-old ndlM mutants. (A) Twinned flowers with two pistils (red arrows). (B) Silique formation from twin flowers of ndlM mutants. The red arrow marks the base of the fused pistils. (C) Shoots of mature ndlM plants show asymmetric silique distribution compared to Col-0 wild-type plants (left). Arrows indicate an abnormally large internode in the ndlM plant. (D) Internodes of ndlM mutants show zones of compaction (arrows). (E) Frequencies of internodal lengths between siliques in ndlM2 mutants compared to Col-0. (F) Comparison of silique development in Col-0 wild-type plants (center), agb1-2 mutants (right) and agb1-2,ndlM2 mutants (left). Downregulation of the NDL genes in the agb1-2 mutant background rescues some of the abnormal agb1-2 silique phenotypes such as shape, angle and internode length.
Figure 6.
Phenotypic analyses of the shoot apical meristem and the axillary meristem in ndlM mutant plants.
(A-E) Field-emission scanning electron microscopy images of the shoot apical meristem (SAM) and the axillary meristem (AM) in ndlM mutants. Developing SAMs were analyzed at various stages of vegetative growth (A) Two-day-old plant. (B) Four-day-old plant. (C) Eight-day-old plant. (D) Fourteen-day-old plant. (E) Three-week-old plant. The arrows in (D) and (E) indicate the positions of the SAMs and the AMs. (F) ndlM plant showing twin rosettes. The white arrows point to each rosette head. LP: leaf primordia, (A-E) Scale bars = 20 µm.
Figure 7.
NDL expression affects auxin transport in the inflorescence stem and local auxin gradients.
(A) Measurement of basipetal auxin transport using [3H]-IAA in inflorescence stems of NDL1 over-expression plants and NDL knock-down (ndlM2A and ndlM2B, two independent ndlM2 lines) plants in comparison to Col-0 wild type and agb1 mutant plants. The mean ± SEM values are based on more than 50 shoots per genotype. Student's t tests are based on differences between wild type and the indicated genotypes. A confidence level of P<0.05 is indicated by an asterisk. (B-E) Histochemical analyses of DR5:GUS expression in NDL knock-down mutants. GUS staining in apical (B) and basal (D) stem sections of ndlM2 mutant lines compared to the corresponding regions of the Col-0 wild-type plants (C and E). (F-I) Histochemical analyses of DR5:GFP expression in NDL1 over-expressing plants (35SNDL1). GUS staining in the apical (F) and basal (G) stem sections of 35SNDL1 lines compared to the corresponding regions in Col-0 wild-type plants (H and I). Scale bars (B to I) = 50 µm, pi = pith. The red arrows and the stars mark the positions of the interfascicular region and the xylem, respectively.
Figure 8.
NDL and AGB1 expression affect relative MAX2 expression levels in flowers.
(A) qRT-PCR analysis to determine NDL- and AGB1-dependent relative MAX2 expression levels. (B) qRT-PCR analysis to determine MAX2-dependent NDL1 expression levels. Reactions were performed in triplicate, and three biological replicates were used. Error bars represent the SE. Student's t test results are based on differences between the wild type and the indicated genotypes and are shown as asterisks: *, P<0.05.
Figure 9.
Proposed model for NDL function in the meristem and inflorescence stems.
The connection between RGS1, GPA1 and AGB is based on data shown in this publication and previously published studies [83]–[86]. NDL1 and AGB1 are positive regulators of basipetal auxin transport (Fig 7A). They also function in auxin-regulated organ formation (Fig 1C and 3) and lateral meristem formation (Figs 2, 5, and 6) by establishing and/or maintaining auxin maxima (Fig 7B to I). Auxin positively regulates AGB1 expression, whereas it has a negative effect on NDL1 stability [40]. Furthermore, auxin increases strigolactone biosynthesis, which subsequently activates MAX2 via SLBPs [5]. Together, NDL1 and AGB1 regulate MAX2 expression (Fig. 8). The scheme does not illustrate the actions of the three NDL proteins. Genetic interactions are represented by straight arrows, biochemical interactions are represented by wavy arrows, and a proposed interaction is represented by a wavy hatched arrow.