Cdi gene in pollen germination


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pollen germination and tube growth: cdi gene...

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  • Pollen germination and tube growth are pivotal to the delivery of the male gametes to the female gametophytes during sexual reproduction of flowering plants. Several biological processes, including dynamic changes in actin cytoskeleton, active membrane trafficking, intracellular signaling and biosynthesis/modification of cell walls, have been identified as involved in pollen tube growth. To accommodate fast tube growth, these tube wall constituents like callose or pectine cellulose and hemicellulose need to be produced and assembled rapidly and precisely. So fer several proteins invoved in this process hav been identified. For e.g.
  • Figure A showing structure of cdi gene with t-dna insertion site in the exonicresion represented as black box. 5` RACE was performed to know no. of exon and introns. T-Dna insertion mutants were identified by pcr of genomic dna using t-dna specific primer. By pcr based method they identified that the the gene was heterozygous. They compared relative expression of cdi in wild type and mutant line utilising RT-PCR. And oberved that the cdi was reduced to half in tdna mutant. Further quantitative RT-PCR analyses showed that Cdi is ubiquitously expressed in many organs/tissues, but that the expression level is significantly higher in pollen
  • To further characterize the expression pattern of Cdi, histochemical analyses were performed. As expected, GUS activity was observed in flowers at late developmental stages, and in pollen grains on stigma (Fig. 2A and B). In isolated stamen, GUS activity was specifically detected in pollen grains (Fig. 2C). GUS activities were also detected in germinated pollen and pollen tubes (Fig. 2E and F). These data suggest that CDI may be involved in pollen development or tube growth.  β-glucuronidasepBI121-Cdi/GUS construct.
  • They suspected that the pollen grains containing mutant allele might be impaired. To confirm this they perfomed crosses and screened progenies on kanamycine selection medium as tdna insert containg k resistant gene. 196 progeny derived from self-pollinated heterozygous cdi-1/+ plants were resistant to kanamycin, while 189 were sensitive, fitting an expectation ratio of 1:1 (v2 = 0.13, p = 0.721). Further reciprocal crosses between the mutant and wild-type plants showed that when cdi-1/+ plants were pollinated with wild type pollen grains, the progeny segregated at a KanR:- KanS ratio of 1:1 (v2 = 0.05, p = 0.824), indicating a 100% transmission efficiency (TE) through female gametophytes (TEf). However, in the reciprocal crosses, where cdi-1/+ pollen grains were used to pollinate wild-type stigmas, no kanamycin resistant progeny were identified (TEm = 0%, Table 1). These results indicate that the mutation in Cdi prevents the genetic transmission from male gametophytes to female gametophytes.
  •  encodes a pectin methylesterase (PME)… QRT1 is expressed in anther tissues shortly after meiosis is complete….. n qrt1 and qrt2 mutants, pectic components of this wall persisted after callose degradation..  failure of pectin degradation in the pollen mother cell wall is associated with tetrad pollen formation in qrt mutants, QRT1 and QRT2, are required for pollen separation during normal development. Quartet 1stained by Alexander staining, indicating that the pollen viabilitywas not affected by the cdi-1 mutation (Fig. 3A and B). Similar resultswere obtained in DAPI staining, where all pollen in tetradscontained two sperms and one vegetative nucleus, suggesting thatnuclear division was not affected by the cdi-1 mutation either(Fig. 3E and F). Further S.E.M. (scanning electron microscopy)inspection of mature pollen (Fig. 3G and H) or sectioning of anthersat different developmental stages (Fig. S1) did not show any obviouschange in cdi-1/+ anthers or pollen grains. These data indicatethat cdi-1 does not affect pollen grain formation.
  • Given that the cdi-1 allele was not transmitted through themale (Table 1), while pollen grain formation was not affected(Figs. 3, S1), we postulated that pollen germination or tube growthmight be affected. As shown in Fig. 4, 65% tetrads from qrt1/qrt1plants germinated with 3 or 4 normal tubes, and 35% with 62(Fig. 4A, E). In contrast, 92% tetrads from cdi-1/+; qrt1/qrt1 contained62 tubes (Fig. 4B, E), while the remaining 8% of the tetradsgerminated with 2 normal tubes plus 1 or 2 aberrant ones with significantlyreduced growth (Fig. 4C and D). Further analyses showedthat the overall germination rate of qrt1/qrt1 pollen was nearlytwice that of cdi-1/+; qrt1/qrt1 (Fig. 4F). These findings are consistentwith the expectation that 50% of these pollen grains produceno or abnormal pollen tubes.
  • Cdi gene in pollen germination

    1. 1. Cdi gene is required for pollengermination and tube growth inArabidopsis.By:- Hong-Mei Li , Hao Chen , Zhong-Nan Yang , Ji-Ming Gong.Li et al., 2012Presentation by : Satya Prakash ChaurasiaDepartment of Botany,University of Delhi, ttp:// chaurasia/65/a76/a25Mob: +919654814497
    2. 2. Introduction• Several biological processes, including dynamic changes in actin cytoskeleton, activemembrane trafficking, intracellular signaling and biosynthesis/modification of cell walls, havebeen identified as involved in pollen tube growth [Fu et al., 2010; Helper et al., 2001].• So far, several proteins involved in biosynthesis and modification of cellulose, hemicelluloseand pectin are known to be required for pollen tube growth [Bernal et al., 2008].– Functional disruption of MGP2 and MGP4 significantly inhibited pollen germination and tube growth[Denge et al., 2010; Lui et al., 2011].– Mutations in CSLD1 and CSLD4 caused a significant reduction in cellulose deposition in tube wallsand a remarkable disorganization of callose, hence disrupting normal pollen germination and tubegrowth [Wang et al., 2011].In the current study , they were aimed to characterize a gene Cdi.MGP> Male Gametophyte DefectiveCSLD>Cellulose Synthase-Like D
    3. 3. (A) Schematic structure of Cdi and T-DNA insertion site. Black box represents exon,open boxes represent UTR regions, dotted line shows intron in 50-UTR. FP: forwardprimer, RP: reverse primer, LBa1: left border primer of the T-DNA insertion. (B) PCRidentification of T-DNA insertion mutants using genomic DNA. (C) Quantitative RT-PCR analysis of Cdi expression in pollen, values are mean S.E., n = 4 replicates.(D) Relative Cdi mRNA expression levels in different tissues. Tubulin gene was usedas an internal control for quantitative assay. Values are mean S.E., n = 3 replicates.Li et al., 2012
    4. 4. Cdi is preferentially expressed in pollen grains and pollen tubes.(A) Histochemical analysis of GUS activity in inflorescence of 35 day-old-plant harboring pBI121-Cdi/GUSconstruct. (B) Close-up imaging of GUS activity in pollen grains on stigma. (C) GUS activities in maturestamen. (D) mature pollen grain. (E and F) Pollen germinated in vitro.Li et al., 2012
    5. 5. Mutation in Cdi prevents the genetic transmission from male gametophytes to femalegametophytes.Li et al., 2012
    6. 6. (A) and (B) Alexander’s stain of pollen from qrt1/qrt1 (A) and cdi-1/+ qrt1/qrt1 (B).(C) and (D) Bright field imaging of pollen from qrt1/qrt1 (C) and cdi-1/+ qrt1/qrt1 (D).(E) and (F) DAPI stain of pollen qrt1/qrt1 (E) and cdi-1/+ qrt1/qrt1 (F). (G) and (H)Representative S.E.M. images of mature pollen from Col-0 (G) and cdi-1/+ (H). Bars= 20 microM in (A–F).Li et al., 2012cdi-1 does not affect pollen grain formation
    7. 7. Mutation in Cdi impairs pollen germination and tube growth.(A) A representative tetrad from the qrt1/qrt1 parental control line showing four normalpollen tubes. (B) Representative tetrad from cdi-1/+ showing two non-germinated pollengrains. (C and D) Representative tetrads from cdi-1/+ showing segregation of normaland aberrant pollen tubes. (E) The graph shows the frequency of tetrads consisting ofone or two tubes (0 + 1 + 2) with normal morphology versus three or four pollen tubes (3+ 4). Pollen tubes were considered aberrant if they failed to grow >50 lm. (F) Germinationrates of pollen grains from qrt1/qrt1 and cdi-1/+;qrt1/qrt1 plants. Scale bars = 50 lm in(A–C) or 25 lm in (D).Li et al., 2012
    8. 8. CDI, What it actually does??• Bioinformatic analyses.– Cdi might encode a putative nucleotide-diphosphosugar transferases superfamilyprotein.• Pfam search– Indicated that CDI may belong to glycosyltransferase (GT) family 8.• DXD motif– extensively found across diverse GT families.Cdi might encode a cytosolic glycosyltransferase
    9. 9. conclusionThey isolated Cdi, encoding a putative glycosyltransferase.Functional analyses established a correlation between cdi-1 andimpaired male transmission. They further showed that Cdi isrequired for pollen germination and tube growth, but does notaffect pollen development or female transmission.
    10. 10. References• Fu, Y. (2010) The actin cytoskeleton and signaling network during pollen tube tip growth. J.Integr. Plant Biol. 52, 131–137.• Hepler, P.K., Vidali, L. and Cheung, A.Y. (2001) Polarized cell growth in higher plants. Annu.Rev. Cell Dev. Biol. 17, 159–187.• Bernal, A.J. et al. (2008) Functional analysis of the cellulose synthase-like genes CSLD1,CSLD2, and CSLD4 in tip-growing Arabidopsis cells. Plant Physiol. 148, 1238–1253.• Liu, X.L. et al. (2011) Male gametophyte defective 4 encodes a rhamnogalacturonan IIxylosyltransferase and is important for growth of pollen tubes and roots in Arabidopsis. PlantJ. 65, 647–660.• [15] Wang, W. et al. (2011) Arabidopsis CSLD1 and CSLD4 are required for cellulosedeposition and normal growth of pollen tubes. J. Exp. Bot. 62, 5161–5177.