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First published online 22 October 2003
doi: 10.1242/dev.00827

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The DUET gene is necessary for chromosome organization and progression during male meiosis in Arabidopsis and encodes a PHD finger protein

Thamalampudi Venkata Reddy1,*, Jagreet Kaur1,*, Bhavna Agashe1,*, Venkatesan Sundaresan2 and Imran Siddiqi1,{dagger}

1 Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad – 500007, India
2 Department of Plant Biology and Agronomy, Life Sciences Addition 1002, University of California, Davis, CA95616, USA



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  		Fig. 1. Wild-type (Ler), duet and revertant plants. (A) Wild-type plant showing normal elongating siliques. (B) duet mutant plant with short siliques. (C) Mutant plant with revertant sector showing elongating silique (arrow). (D) Wild-type flower with long anther filaments and plentiful pollen. (E) duet flower with short filaments and anthers lacking pollen. (F) Wild-type cDNA and derived amino acid sequence of DUET near the site of the Ds insertion. The duet mutant sequence shows a 8 bp duplication (bold underlined). The two excision alleles have a 7 and 8 bp footprint respectively (bold).

 


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  		Fig. 2. Anther and pollen development in wild type and duet. Plastic cross-sections of anthers. (A,D,G) Wild type; (B,C,E,F,H,I) duet. (A,B) Anthers with pollen mother cells at stage 5, all the layers of the anther are present in both genotypes. (C) duet microspore mother cell at meiosis; pollen mother cells (PMCs) surrounded by a layer of callose. (D) Stage 7 anther showing tetrads held together by a layer of callose. (E) duet anther with dyads, triads, tetrads: products of a defective meiosis. (F) Products of aberrant meiosis separate out, enlarge and undergo nuclear division (arrowheads). (G) Stage 12 anther containing mature pollen. (H) Enlarged microspore-like cells (arrowheads) with a single nucleus and lacking exine. (I) Late stage anther showing empty locules. CL, callose layer; E, epidermis; En, endothecium; Ml, middle layer; PMC, pollen mother cell; T, tapetum; Tds, tetrads. Scale bars: 25 µm (A,B,C,F,G,I) and 12.5 µm (D,H,E).

 


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  		Fig. 3. Stages of male meiosis and pollen development in wild type and duet. Optical sections of cleared anthers viewed under DIC optics. (A-D) Wild type; (E-K) duet. (A,E) Stage 5 anthers with pollen mother cells (arrowheads). (B) Stage 7 anther containing tetrads (arrowhead). (C) Microspores released from the tetrad generate an exine wall and become vacuolated (arrowhead). (D) Mature pollen. (F,G) Dyads (arrowhead in F) formed after a defective meiosis. (H,I) Cells of the dyad separate and enlarge (arrowhead in H). (J,K) The microspore-like cells released from the dyad enlarge and undergo nuclear division to form 2-3 nucleate cells (arrowhead in J), which later degenerate. Scale bars: 12.5 µm.

 


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  		Fig. 4. (A) Schematic representation of the DUET gene. The exons are indicated with black boxes. Arrows indicate the primers used for cDNA isolation and expression analysis. Coordinates are with respect to BAC F15E12. (B) The predicted sequence of DUET protein. The putative nuclear localization signal is in bold. The region showing homology to DYAD is underlined and the PHD-finger domain is boxed. The inverted triangle after amino acid 550 indicates the Ds transposon insertion site. (C) Analysis of DUET expression by RT-PCR. Expression of the DUET gene was examined in wild type (Wt) leaves (Lea), inflorescence (Inf) and duet mutant inflorescence by amplifying the cDNA synthesized from poly(A)+ mRNA. The shift in the size of the amplicon can be observed when genomic DNA (gen) was used as template. The constitutive GAPC gene was used as the normalization control. (D) Comparison of DUET and DYAD cDNA in pistils.

 


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  		Fig. 5. Expression of DUET in male meiocytes: RNA in situ hybridization of DUET antisense RNA to sections of flower buds. (A) Expression is first seen in sporogenous cells at anther stage 4. (B,C) Maximal expression is seen in microsporocytes at anther stage 5. (D) Anther stage 6, meiotic cells. (E,F) No expression is seen in the female meiocyte at stage 2-3 corresponding to pre-meiosis. (E) Antisense; (F) sense control. Scale bars: 50 µm.

 


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  		Fig. 6. Chromosome analysis in spreads of male meiocytes in wild type and duet. (A-J) Wild type; (K-W) duet. (A,K) Chromosomes first become visible as elongated strands during leptotene. (B,L) Synapsis takes place during zygotene. (C,M) Synapsis is complete at pachytene and chromosomes have a shorter and thicker appearance. (D) Diplotene stage, when bivalents have undergone partial decondensation. (E) Late diakinesis showing five pairs of chromosomes with chiasmata at their ends. (F) Metaphase I stage showing five condensed bivalents arranged on a metaphase plate. (G) Telophase I: five chromosomes at each end are separated by an organelle band. (H) Metaphase II. (I) Anaphase II. (G-I) Arrows indicate the densely compacted organelle band. (J) Telophase II where four groups of five chromosomes each have separated. (N) First apparent visible defect in duet at diplotene. Chromosomes start to look diffuse and two bivalents have undergone partial desynapsis (arrowheads). (O) A more severe form of desynapsis can be observed in the majority of bivalents. (P,Q) Disorganized diakinesis in duet with diffuse chromosomes including the centromeric region. (R,S,T) Metaphase I. (U) Anaphase I. (V) Defective anaphase I stage with fragmented chromosome and laggards. (W) Telophase I. The organelle band is absent. Scale bars: 12.5 µm.

 


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  		Fig. 7. Quantitation of different stages of male meiosis in wild type and duet. (White bars) Wild type; (Black bars) duet; leptotene (L), zygotene (Z), pachytene (P), diplotene (DP), diakinesis (DI), metaphase I (M-I), anaphase I (A-I), telophase I (T-I), metaphase II (M II), anaphase II (A-II), telophase (T-II) and tetrads (TD). Note the large increase in the proportion of metaphase I cells in duet.

 


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  		Fig. 8. Interactions between duet and dyad during male meiosis and pollen development. Optical sections of cleared anthers viewed under DIC optics. (A-D) duet dyad double mutant; (E-L) duet/+ dyad/dyad. (A,E) Normal-looking meiocytes (arrowheads). (B) Defective microspores lacking exine (arrowhead). (C) Binucleate spores. (D) Late stage defective binucleate and multinucleate spores (arrow and arrowhead, respectively) surrounded by an uneven wall resembling the exine; not observed in the duet single mutant. (F) Dyad (arrowhead). (G) Undivided triads (arrowhead). (H) Round spores containing 1-3 nuclei. (I) Normal-looking tetrads (arrowhead). (J) Normal-looking developing pollen. (K) Abnormal multinucleate structures undergoing shrinkage (arrowhead). (L) Shrunken pollen. Scale bar: 12.5 µm.

 


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  		Fig. 9. Interactions between duet and dyad: meiotic chromosome stages. (A-J) duet/+ dyad/dyad. (K-N,W) duet dyad double mutant. (O-Q) duet/+ heterozygote. (R-T,V) dyad mutant. (A) Early zygotene. (B) Pachytene stage showing thickened but irregular chromosomes. (C) Diakinesis. Five bivalents are visible. (D) Aberrant diakinesis in which chromosomes have desynapsed to form ten univalents. (E) Extreme diakinesis in which both synapsis and sister chromatid cohesion have been lost to yield single chromatids. (F) Early anaphase 1 undergoing mixed segregation in which both univalents and bivalents are involved. (G) Late anaphase 1 showing approximately equal separation of chromosomes. Eight to ten chromosomes are present at each pole indicating an equal division. (H) Telophase 1. Equal division. Ten chromosomes are present at each pole. (I) Telophase 1. Unequal division. (J) Dyad formed after unequal division. (K) Zygotene. (L) Diakinesis involving 2 bivalents and 6 univalents. (M) Extreme diakinesis containing mostly single chromatids. (N) Prometaphase 1 having eight to ten thick diffuse chromosomes. (O,R) Normal diakinesis. (P,S) Metaphase 1. (Q,T) Telophase 1. (U,V) Cleared ovules of dyad (U) and duet dyad (V). (W) Metaphase 1. Scale bars: 12.5 µm (A-T,W) and 25 µm (U,V).

 




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