The mapping of functional genes plays an important role in studies of genome structure, function, and evolution, as well as allowing gene cloning and marker-assisted selection to improve agriculturally important traits. Simple sequence repeats (SSRs) developed from expressed sequence tags (ESTs), EST-SSR (eSSR), can be employed as putative functional marker loci to easily tag corresponding functional genes. In this paper, 2218 eSSRs, 1554 from G. raimondii-derived and 754 from G. hirsutum-derived ESTs, were developed and used to screen polymorphisms to enhance our backbone genetic map in allotetraploid cotton. Of the 1554 G. raimondii-derived eSSRs, 744 eSSRs were able to successfully amplify polymorphisms between our two mapping parents, TM-1 and Hai7124, presenting a polymorphic rate of 47.9%. However, only a 23.9% (159/754) polymorphic rate was produced from G. hirsutum-derived eSSRs. No relationship was observed between the level of polymorphism, motif type, and tissue origin, but the polymorphism appeared to be correlated with repeat type. After integrating these new eSSRs, our enhanced genetic map consists of 1790 loci in 26 linkage groups and covers 3425.8 cM with an average intermarker distance of 1.91 cM. This microsatellite-based, gene-rich linkage map contains 71.96% functional marker loci, of which 87.11% are eSSR loci. There were 132 duplicated loci bridging 13 homeologous At/Dt chromosome pairs. Two reciprocal translocations after polyploidization between A2 and A3, and between A4 and A5, chromosomes were further confirmed. A functional analysis of 975 ESTs producing 1122 eSSR loci tagged in the map revealed that 60% had clear BLASTX hits (,1e À10 ) to the Uniprot database and that 475 were associated mainly with genes belonging to the three major gene ontology categories of biological process, cellular component, and molecular function; many of the ESTs were associated with two or more category functions. The results presented here will provide new insights for future investigations of functional and evolutionary genomics, especially those associated with cotton fiber improvement.

Ca(2+) and calmodulin (CaM) have been shown to play an important role in abscisic acid (ABA)-induced antioxidant defense. However, it is unknown whether Ca(2+)/CaM-dependent protein kinase (CCaMK) is involved in the process. In the present study, the role of rice CCaMK, OsDMI3, in ABA-induced antioxidant defense was investigated in leaves of rice (Oryza sativa) plants. Treatments with ABA, H(2)O(2), and polyethylene glycol (PEG) induced the expression of OsDMI3 and the activity of OsDMI3, and H(2)O(2) is required for the ABA-induced increases in the expression and the activity of OsDMI3 under water stress. Subcellular localization analysis showed that OsDMI3 is located in the nucleus, the cytoplasm, and the plasma membrane. The analysis of the transient expression of OsDMI3 in rice protoplasts and the RNA interference (RNAi) silencing of OsDMI3 in rice protoplasts showed that OsDMI3 is required for ABA-induced increases in the expression and the activities of superoxide dismutase (SOD) and catalase (CAT). Further, the oxidative damage induced by higher concentrations of PEG and H(2)O(2) was aggravated in the mutant of OsDMI3. Moreover, the analysis of the RNAi silencing of OsDMI3 in protoplasts and the mutant of OsDMI3 showed that higher levels of H(2)O(2) accumulation require OsDMI3 activation in ABA signaling, but the initial H(2)O(2) production induced by ABA is not dependent on the activation of OsDMI3 in leaves of rice plants. Our data reveal that OsDMI3 is an important component in ABA-induced antioxidant defense in rice.

Nitric oxide (NO), hydrogen peroxide (H2O2), and calcium (Ca2+)/calmodulin (CaM) are all required for abscisic acid (ABA)-induced antioxidant defence. Ca2+/CaM-dependent protein kinase (CCaMK) is a strong candidate for the decoder of Ca2+ signals. However, whether CCaMK is involved in ABA-induced antioxidant defence is unknown. The results of the present study show that exogenous and endogenous ABA induced increases in the activity of ZmCCaMK and the expression of ZmCCaMK in leaves of maize. Subcellular localization analysis showed that ZmCCaMK is located in the nucleus, the cytoplasm, and the plasma membrane. The transient expression of ZmCCaMK and the RNA interference (RNAi) silencing of ZmCCaMK analysis in maize protoplasts revealed that ZmCCaMK is required for ABA-induced antioxidant defence. Moreover, treatment with the NO donor sodium nitroprusside (SNP) induced the activation of ZmCCaMK and the expression of ZmCCaMK. Pre-treatments with an NO scavenger and inhibitor blocked the ABA-induced increases in the activity and the transcript level of ZmCCaMK. Conversely, RNAi silencing of ZmCCaMK in maize protoplasts did not affect the ABA-induced NO production, which was further confirmed using a mutant of OsCCaMK, the homologous gene of ZmCCaMK in rice. Moreover, H2O2 was also required for the ABA activation of ZmCCaMK, and pre-treatments with an NO scavenger and inhibitor inhibited the H2O2-induced increase in the activity of ZmCCaMK. Taken together, the data clearly suggest that ZmCCaMK is required for ABA-induced antioxidant defence, and H2O2-dependent NO production plays an important role in the ABA-induced activation of ZmCCaMK.

In rice, the Ca 2+ /calmodulin (CaM)-dependent protein kinase (CCaMK) OsDMI3 has been shown to be required for abscisic acid (ABA)-induced antioxidant defence. However, it is not clear how OsDMI3 participates in this process in rice. In this study, the cross-talk between OsDMI3 and the major ABA-activated MAPK OsMPK1 in ABA-induced antioxidant defence was investigated. ABA treatment induced the expression of OsDMI3 and OsMPK1 and the activities of OsDMI3 and OsMPK1 in rice leaves. In the mutant of OsDMI3, the ABA-induced increases in the expression and the activity of OsMPK1 were substantially reduced. But in the mutant of OsMPK1, the ABA-induced increases in the expression and the activity of OsDMI3 were not affected. Pretreatments with MAPKK inhibitors also did not affect the ABA-induced activation of OsDMI3. Further, a transient expression analysis in combination with mutant analysis in rice protoplasts showed that OsMPK1 is required for OsDMI3-induced increases in the activities of antioxidant enzymes and the production of H2O2. Our data indicate that there exists a cross-talk between OsDMI3 and OsMPK1 in ABA signalling, in which OsDMI3 functions upstream of OsMPK1 to regulate the activities of antioxidant enzymes and the production of H2O2 in rice.