We took “input”samples of cleared cell lysates before we added beads

loids and their very narrow genetic base, therefore allows highly accurate gene assemblies to be generated from mixed ESTs from amongst the four different Gossypium species for which extensive ESTs have been lodged in GenBank. After blasting the translated cotton EST databases with PME protein sequences from Arabidopsis and assembling those hits into contigs, we were able to identify at least 33 distinct expressed cotton PME genes from over 520 EST sequences derived from a variety of cotton tissues and species. Subsequently, a draft assembly of the diploid G. raimondii genome was released and this allowed a more extensive survey of Gossypium PMEs using the same search strategy. Although 102 gene models were identified in G. raimondii with a Blast hit to AtPME2 or AtPME31 and/or our 33 identified EST contigs, 21 of those were characterised as PME inhibitors by the NCBI Conserved Domain Database Search tool and lacked a PME catalytic domain. This expanded the number of potential cotton PMEs from 33 to a total of 81. These PME genes were distributed across all 13 chromosomes of G. raimondii with several being clustered in groups of two, three or four genes in tandem order suggesting they have evolved through gene duplications and later divergence. The gene models encoded predicted PME proteins ranging from as little as 120 amino acids to one as large as 1262 aa, although generally they were less than 600 aa. The predicted PME inhibitor proteins were usually around 200 aa. Amongst the predicted PMEs, the smaller proteins generally only contained a single conserved PME catalytic domain when queried in the CDD, whereas the larger proteins contained a conserved N-terminal PMEI domain and a C-terminal PME catalytic domain. A phylogenetic analysis of the encoded proteins of the 102 putative G. raimondii PME and PMEIs along with the 66 known and putative PMEs from Arabidopsis clustered the cotton genes dispersed among the four different groups of plant PMEs previously defined in, with a separated clade that included all the PMEI only proteins. As they specifically bind to the active site of plant PMEs, PMEI proteins are thought to play important roles in modulating PME activity in muro during growth and development, but they have also been implicated in host defense responses to pathogens. Their possible expression and functions in cotton fibres remains to be explored, so they are not considered further here. Relative to Arabidopsis the greatest expansion in PME genes appears to have been in the group 4 PMEs that contained over 64% more members than in Arabidopsis, whereas as the other three groups all had similar numbers in the two species. Although most cotton PMEs have a related Arabidopsis protein they tend to group more closely with each other in small sub-clades suggesting they may have specialised to new functions in cotton and we were particularly interested in those that might have a novel function in cotton seed fibres that are relatively unique plant seed epidermal trichomes not found in Arabidopsis. Based on the presence of matching ESTs in GenBank many of the cotton PME genes are expressed in young flower buds, developing ovules 26976569 and in some cases cotton fibres, although relatively few had abundant ESTs from cDNA libraries made exclusively from only fibres. Just five of the cotton PME genes, all PMEI-PME types, 518303-20-3 appeared to be expressed abundantly in isolated cotton fibres 9226999 and these were studied in more detail in the two tetraploid cot