BackgroundAbscission is a mechanism by which plants shed entire organs in response to both developmental and environmental signals. Arabidopsis thaliana, in which only the floral organs abscise, has been used extensively to study the genetic, molecular and cellular processes controlling abscission. Abscission in Arabidopsis requires two genes that encode functionally redundant receptor-like protein kinases, HAESA (HAE) and HAESA-LIKE 2 (HSL2). Double hae hsl2 mutant plants fail to abscise their floral organs at any stage of floral development and maturation.ResultsUsing RNA-Seq, we compare the transcriptomes of wild-type and hae hsl2 stage 15 flowers, using the floral receptacle which is enriched for abscission zone cells. 2034 genes were differentially expressed with a False Discovery Rate adjusted p < 0.05, of which 349 had two fold or greater change in expression. Differentially expressed genes were enriched for hydrolytic, cell wall modifying, and defense related genes. Testing several of the differentially expressed genes in INFLORESCENCE DEFICIENT IN ABSCISSION (ida) mutants shows that many of the same genes are co-regulated by IDA and HAE HSL2 and support the role of IDA in the HAE and HSL2 signaling pathway. Comparison to microarray data from stamen abscission zones show distinct patterns of expression of genes that are dependent on HAE HSL2 and reveal HAE HSL2- independent pathways.ConclusionHAE HSL2-dependent and HAE HSL2-independent changes in genes expression are required for abscission. HAE and HSL2 affect the expression of cell wall modifying and defense related genes necessary for abscission. The HAE HSL2-independent genes also appear to have roles in abscission and additionally are involved in processes such as hormonal signaling, senescence and callose deposition.

SummaryMcCDPK1 is a salinity-and drought-induced calcium-dependent protein kinase (CDPK) isolated from the common ice plant, Mesembryanthemum crystallinum. A yeast two-hybrid experiment was performed, using full-length McCDPK1 and truncated forms of McCDPK1 as baits, to identify interacting proteins. A catalytically impaired bait isolated a cDNA clone encoding a novel protein, CDPK substrate protein 1 (CSP1). CSP1 interacted with McCDPK1 in a substrate-like fashion in both yeast two-hybrid assays and wheat germ interaction assays. Furthermore, McCDPK1 was capable of phosphorylating CSP1 in vitro in a calcium-dependent manner. Our results demonstrate that the use of catalytically impaired and unregulated CDPKs with the yeast two-hybrid system can accelerate the discovery of CDPK substrates. The deduced CSP1 amino acid sequence indicated that it is a novel member of a class of pseudoresponse regulator-like proteins that have a highly conserved helix±loop±helix DNA binding domain and a C-terminal activation domain. McCDPK1 and CSP1 co-localized to nuclei of NaCl-stressed ice plants. Csp1 transcript accumulation was not regulated by NaCl or dehydration stress. Our results strongly suggest that McCDPK1 may regulate the function of CSP1 by reversible phosphorylation.

Drought-triggered abscission is a strategy used by plants to avoid the full consequences of drought; however, it is poorly understood at the molecular genetic level. Here, we show that Arabidopsis (Arabidopsis thaliana) can be used to elucidate the pathway controlling drought-triggered leaf shedding. We further show that much of the pathway regulating developmentally timed floral organ abscission is conserved in regulating drought-triggered leaf abscission. Gene expression of HAESA (HAE) and INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) is induced in cauline leaf abscission zones when the leaves become wilted in response to limited water and HAE continues to accumulate in the leaf abscission zones through the abscission process. The genes that encode HAE/HAESA-LIKE2, IDA, NEVERSHED, and MAPK KINASE4 and 5 are all necessary for drought-induced leaf abscission. Our findings offer a molecular mechanism explaining drought-triggered leaf abscission. Furthermore, the ability to study leaf abscission in Arabidopsis opens up a new avenue to tease apart mechanisms involved in abscission that have been difficult to separate from flower development as well as for understanding the mechanistic role of water and turgor pressure in abscission.

A salinity and dehydration stress-responsive calcium-dependent protein kinase (CDPK) was isolated from the common ice plant (Mesembryanthemum crystallinum; McCPK1). McCPK1 undergoes myristoylation, but not palmitoylation in vitro. Removal of the N-terminal myristate acceptor site partially reduced McCPK1 plasma membrane (PM) localization as determined by transient expression of green fluorescent protein fusions in microprojectile-bombarded cells. Removal of the N-terminal domain (amino acids 1-70) completely abolished PM localization, suggesting that myristoylation and possibly the N-terminal domain contribute to membrane association of the kinase. The recombinant, Escherichia coli-expressed, full-length McCPK1 protein was catalytically active in a calcium-dependent manner (K 0.5 5 0.15 mM). Autophosphorylation of recombinant McCPK1 was observed in vitro on at least two different Ser residues, with the location of two sites being mapped to Ser-62 and Ser-420. An Ala substitution at the Ser-62 or Ser-420 autophosphorylation site resulted in a slight increase in kinase activity relative to wild-type McCPK1 against a histone H1 substrate. In contrast, Ala substitutions at both sites resulted in a dramatic decrease in kinase activity relative to wild-type McCPK1 using histone H1 as substrate. McCPK1 undergoes a reversible change in subcellular localization from the PM to the nucleus, endoplasmic reticulum, and actin microfilaments of the cytoskeleton in response to reductions in humidity, as determined by transient expression of McCPK1-green fluorescent protein fusions in microprojectile-bombarded cells and confirmed by subcellular fractionation and western-blot analysis of 63 His-tagged McCPK1.Calcium is a ubiquitous and pivotal second messenger in the signal transduction networks that plants use to respond to a wide variety of physiological stimuli. Cytosolic Ca 21 fluctuations have been observed in response to a number of stimuli, including red light, abscisic acid (ABA), GA, drought, hyperand hypo-osmotic stress, ionic stress, touch, cold, heat shock, oxidative stress, fungal elicitors, and nodulation factors (Sanders et al., 2002 (Harmon et al., , 2001Hrabak, 2000;Hrabak et al., 2003).CDPKs are composed of a single polypeptide chain with a catalytic kinase domain at the N terminus, an intervening junction domain, and a calmodulin-like domain at the C terminus containing up to four functional Ca 21 -binding EF hands. The junction domain acts as an autoinhibitor in a pseudosubstrate fashion (Harper et al., 1994;Vitart et al., 2000;Weljie et al., 2000). Binding of Ca 21 to the calmodulin-like domain results in a conformational change leading to the release of the autoinhibitor domain from the active site and kinase activation (Harmon et al., 1994;Harper et al., 1994). Many CDPKs also have additional Nterminal leader and C-terminal domains composed of highly variable amino acid sequences. AtCPK25 and some CDPK-related kinases possess degenerate calmodulin domains (Lindzen and Choi, 1995;Zhang and Lu, 2003) and appa...