Drought and salinity are two major limiting factors in crop productivity. One way to reduce crop loss caused by drought and salinity is to increase the solute concentration in the vacuoles of plant cells. The accumulation of sodium ions inside the vacuoles provides a 2-fold advantage: (i) reducing the toxic levels of sodium in cytosol; and (ii) increasing the vacuolar osmotic potential with the concomitant generation of a more negative water potential that favors water uptake by the cell and better tissue water retention under high soil salinity. The success of this approach was demonstrated in several plants, where the overexpression of the Arabidopsis gene AtNHX1 that encodes a vacuolar sodium/proton antiporter resulted in higher plant salt tolerance. Overexpression of AtNHX1 increases sodium uptake in vacuoles, which leads to increased vacuolar solute concentration and therefore higher salt tolerance in transgenic plants. In an effort to engineer cotton for higher drought and salt tolerance, we created transgenic cotton plants expressing AtNHX1. These AtNHX1-expressing cotton plants generated more biomass and produced more fibers when grown in the presence of 200 mM NaCl in greenhouse conditions. The increased fiber yield was probably due to better photosynthetic performance and higher nitrogen assimilation rates observed in the AtNHX1-expressing cotton plants as compared with wild-type cotton plants under saline conditions. Furthermore, the field-grown AtNHX1-expressing cotton plants produced more fibers with better quality, indicating that AtNHX1 can indeed be used for improving salt stress tolerance in cotton.

The Arabidopsis gene GF14 lambda that encodes a 14-3-3 protein was introduced into cotton plants to explore the physiological roles that GF14 lambda might play in plants. The expression level of GF14 lambda under the control of the cauliflower mosaic virus 35S promoter varied in transgenic cotton plants, and lines that expressed GF14 lambda demonstrated a "stay-green" phenotype and improved water-stress tolerance. These lines wilted less and maintained higher photosynthesis than segregated non-transgenic control plants under water-deficit conditions. Stomatal conductance appears to be the major factor for the observed higher photosynthetic rates under water-deficit conditions. The stomatal aperture of transgenic plants might be regulated by GF14 lambda through some transporters such as H(+)-ATPase whose activities are controlled by their interaction with 14-3-3 proteins. However, since 14-3-3 proteins interact with numerous proteins in plant cells, many metabolic processes could be affected by the GF14 lambda overexpression. Whatever the mechanisms, the traits observed in the GF14 lambda-expressing cotton plants are beneficial to crops under certain water-deficit conditions.

A cytosolic sperm protein(s), referred to as sperm factor (SF), is delivered into eggs by the sperm during mammalian fertilization to induce repetitive increases in the intracellular concentration of free Ca2+ ([Ca2+]i) that are referred to as [Ca2+]i oscillations. [Ca2+]i oscillations are essential for egg activation and early embryonic development. Recent evidence shows that the novel sperm-specific phospholipase C (PLC), PLCzeta, may be the long sought after [Ca2+]i oscillation-inducing SF. Here, we demonstrate the complete extraction of SF from porcine sperm and show that regardless of the method of extraction a single molecule/complex appears to be responsible for the [Ca2+]i oscillation-inducing activity of these extracts. Consistent with this notion, all sperm fractions that induced [Ca2+]i oscillations, including FPLC-purified fractions, exhibited high in vitro PLC activity at basal Ca2+ levels (0.1-5 microM), a hallmark of PLCzeta. Notably, we detected immunoreactive 72-kDa PLCzeta in an inactive fraction, and several fractions capable of inducing oscillations were devoid of 72-kDa PLCzeta. Nonetheless, in the latter fractions, proteolytic fragments, presumably corresponding to cleaved forms of PLCzeta, were detected by immunoblotting. Therefore, our findings corroborate the hypothesis that a sperm-specific PLC is the main component of the [Ca2+]i oscillation-inducing activity of sperm but provide evidence that the presence of 72-kDa PLCzeta does not precisely correspond with the Ca2+ releasing activity of porcine sperm fractions.

CHIP proteins are E3 ubiquitin ligases that promote degradation of Hsp70 and Hsp90 substrate proteins through the 26S proteasome in animal systems. A CHIP-like protein in Arabidopsis, AtCHIP, also has E3 ubiquitin ligase activity and has important roles to play under conditions of abiotic stress. In an effort to study the mode of action of AtCHIP in plant cells, proteins that physically interact with it were identified. Like its animal orthologs, AtCHIP interacts with a unique class of ubiquitin-conjugating enzymes (UBC or E2) that belongs to the stress-inducible UBC4/5 class in yeast. AtCHIP also interacts with other proteins, including an A subunit of protein phosphatase 2A (PP2A). This PP2A subunit appears to be a substrate of AtCHIP, because it can be ubiquitylated by AtCHIP in vitro and because the activity of PP2A is increased in AtCHIP-overexpressing plants in the dark or under low-temperature conditions. Unlike the rcn1 mutant, that has reduced PP2A activity due to a mutation in one of the A subunit genes of PP2A, AtCHIP-overexpressing plants are more sensitive to ABA treatment. Since PP2A was previously shown to be involved in low-temperature responses in plants, the low-temperature-sensitive phenotype observed in AtCHIP-overexpressing plants might be partly due to the change in PP2A activity. These data suggest that the E3 ubiquitin ligase AtCHIP may function upstream of PP2A in stress-responsive signal transduction pathways under conditions of low temperature or in the dark.

We reported previously that egg membrane rafts serve as a subcellular microdomain for sperm-dependent tyrosine kinase signaling in Xenopus fertilization. Moreover, we demonstrated that raft-associated Src tyrosine kinase was activated by sperm in vitro. Here we show that egg rafts incubated with sperm or hydrogen peroxide (H 2 O 2 ) can promote Src-dependent phosphorylation of phospholipase C␥ (PLC␥) and transient calcium release in the extracts of unfertilized Xenopus eggs. In vivo egg activation by sperm or H 2 O 2 also promotes tyrosinephosphorylation and raft-translocalization of PLC␥. Immunodepletion of PLC␥ from the egg extracts inhibits the raft-dependent calcium release. Rafts prepared from H 2 O 2 -activated eggs also promote Src-dependent dephosphorylation of p42 mitogen-activated protein kinase and cell cycle transition from metaphase II to interphase in egg extracts. PLC␥ phosphorylation and calcium release in egg extracts can be promoted by rafts prepared from COS-7 cells expressing the Xenopus Src gene. These results demonstrate that the signaling events elicited by fertilization in Xenopus eggs can be reconstituted in vitro. The development of such experimental platforms will allow us to dissect the molecular mechanism of sperm-dependent activation of raft-associated Src and subsequent up-regulation of PLC␥ and egg activation machinery in Xenopus eggs.

In mammals, weaning of neonates and subsequent milk stasis initiates removal of the secretory epithelium of the mammary gland by apoptosis. The p53 tumor suppressor gene is induced rapidly following weaning of neonates, but its role in the process of involution has not been de®ned. Therefore, experiments were performed to identify the cell types in which the p53 gene is expressed during involution and determine the consequences of its absence in BALB/c-p53 null mice. Both p53 mRNA and protein were detected in the mammary epithelium within 48 h following weaning and resulted in an eightfold increase in levels of p21 WAF1 mRNA. Induction of p21 WAF1mRNA was absent in BALB/c-p53 null mice, and therefore, was shown to be p53-dependent. The BALB/cp53 null mice exhibited delayed involution of the mammary epithelium, as measured by 60% greater epithelial area compared to BALB/c-p53 wt mice through 5 days postweaning. The delay was transient with no dierences being apparent at 7 days post-weaning. Expression of the stromal protease stromelysin-1 was unaected by the absence of p53 suggesting that stromal responses were intact. These data demonstrate that p53 participates in the ®rst stage of involution initiated by the epithelium itself, but does not aect the second phase during which stromal proteases are induced.

SUMMARY Xenopus oocyte death is partly controlled by the apoptotic initiator, caspase-2. We reported previously that oocyte nutrient depletion activates caspase-2 upstream of mitochondrial cytochrome c release. Conversely, nutrient-replete oocytes inhibit caspase-2 via S135 phosphorylation catalyzed by calcium/calmodulin-dependent protein kinase II. We now show that caspase-2 phosphorylated at S135 binds 14-3-3ζ, thus preventing caspase-2 dephosphorylation. Moreover, we determined that S135 dephosphorylation is catalyzed by protein phosphatase-1, which directly binds caspase-2. Although caspase-2 dephosphorylation is responsive to metabolism, neither PP1 activity nor binding is metabolically regulated. Rather, release of 14-3-3ζ from caspase-2 is controlled by metabolism and allows for caspase-2 dephosphorylation. Accordingly, a caspase-2 mutant unable to bind 14-3-3ζ is highly susceptible to dephosphorylation. Although this mechanism was initially established in Xenopus, we now demonstrate similar control of murine caspase-2 by phosphorylation and 14-3-3 binding in mouse eggs. These findings provide an unexpected evolutionary link between 14-3-3 and metabolism in oocyte death.

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