NADP Malic Enzymes in C3 and C4 Flaveria Species

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The genus Flaveria is unusual in containing C₃, C₄, and C₃-C₄ intermediate species. This diversity makes Flaveria an interesting subject for studying the molecular events that have accompanied the evolutionary transition from C₃ to C₄ photosynthesis. In the most common C₄ pathway for C fixation, NADP-malic enzyme (NADP-ME) is involved in decarboxylating malate in the chloroplasts of bundle sheath cells. Isoforms of plastidic NADP-ME are encoded by two genes in all species of Flaveria, including C₃, C₄, and C₃-C₄ intermediate types. In this issue, Lai, Wang, and Nelson (pp. 125-139) report that only one of these genes (ChlME1) encodes for the isoform involved in C₄ photosynthesis. A comparison of the expression patterns of ChlMe1 and ChlME2 genes in developing leaves of C₃ and C₄ Flaveria species revealed that in C₄ species, ChlMe1 is expressed non-specifically early in leaf development and becomes bundle sheath-specific as leaves mature (Fig. 1). In C₃ species, however, ChlMe1 is only transiently expressed early in leaf development. In contrast, ChlMe2 expression occurs only transiently during chloroplast development in both C₃ and C₄ species, possibly serving to provide a burst of NADPH and pyruvate for protein and lipid synthesis during chloroplast biogenesis. These results indicate that during the course of C₄ evolution, the expression pattern of ChlMe2 remained constant, while the expression pattern of ChlMe1 changed markedly.

View larger version (142K): [in this window] [in a new window]   Figure 1.   Expression of one of the forms of chloroplastic NADP-ME in the bundle sheath cells of the mature leaves of a C4-type Flaveria species indicates its involvement in C4 photosynthesis.

A companion paper by Lai, Tausta, and Nelson (pp. 140-149) examines the role of cytosolic NADP-ME in Flaveria. They show that the gene CytMe encodes for cytosolic NADP-ME in all Flaveria species regardless of the species' mode of photosynthesis. Based on the expression pattern of CytMe, the authors propose that cytosolic NADP-ME has several distinct roles in plants, including the supplying of NADPH for cytosolic metabolism, the balancing of cellular pH in illuminated leaves, and in providing reducing agents and carbon metabolites during wound repair. CytMe transcripts of different size appear to be involved in these three different processes.

	Nitric Oxide: A Key Link in Abscisic Acid-Induced Stomatal Closure

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The process by which abscisic acid (ABA) induces stomatal closure has been intensively studied, but despite great effort, our understanding of the ABA signal transduction mechanism in guard cells is far from clear. Perhaps some important pieces of the puzzle are provided by Neill et al. (pp. 13-16), who present pharmacological evidence that nitric oxide (NO) plays a critical role in ABA-induced stomatal closure in pea (Pisum sativum). NO causes stomatal closure, and both inhibitors of NO synthesis and NO scavengers block ABA-induced stomatal closure. Neill et al. also employed diaminofluorescein diacetate (DAF-2 DA), a fluorescent indicator probe to visualize NO levels in guard cells under various pharmacological treatments. The application of ABA increased DAF-2 DA fluorescence in pea guard cells, and this increase was prevented by pretreatment with either a NO scavenger or an inhibitor of NO synthesis (Fig. 2). Because NO signaling commonly involves the production of the second messengers cyclic GMP and cADP-Rib, the authors also studied the respective effects of a specific inhibitor of NO-sensitive guanylate cyclase and an antagonist of cADP-Rib on stomatal aperture. Neither of these inhibitors alone had an effect on stomatal aperture, but both inhibited ABA- and NO-induced stomatal closure. The authors propose that NO is a key link in ABA-induced stomatal closure, and that ABA- and NO-induced stomatal closures require the synthesis and action of cyclic GMP and cADP-Rib.

View larger version (57K): [in this window] [in a new window]   Figure 2.   The fluorescent indicator probe DAF-2 DA reveals NO synthesis in pea guard cells under different pharmacological conditions: a, control; b, ABA; c, ABA and NO scavenger; d, ABA and NO synthase inhibitor.

	New Light on the Functions of Phytochromes

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The perception of red (R) and far-red (FR) light by various phytochromes affects the growth and development of a plant throughout its life. In Arabidopsis, phytochrome is a small gene family consisting of five members, PHYA through PHYE. Individual phytochrome family members have both partially overlapping and distinct functions. In the case of the photoregulation of hypocotyl elongation, phyB mediates the classic R/FR photoreversible low fluence response (LFR). In contrast, phyA mediates two other types of responses: the high irradiance response (HIR) that requires sustained exposure to FR, and the very-low-fluence response (VLFR) that is mediated by brief exposures to radiation between 300 and 780 nm. In this issue, Luccioni et al. (pp. 173-181) report on their studies of the relative magnitudes of HIR, VLFR and LFR responses in different accessions of Arabidopsis. Their analysis reveals a significant negative correlation between VLFR and LFR or HIR. The authors also provide tantalizing evidence that brassinosteroids may be part of the "switch" mechanism that adjusts plant sensitivity to light by means of these different phytochrome responses. A mutant that displays an enhanced VLFR but reduced HIR and LFR was found to be allelic to a brassinosteroid biosynthesis mutant. The enhancement of VLFR by this mutation was lost in seedlings not expressing functional phyA. The authors suggest that brassinosteroids may play a role in fine tuning a plant's repertoire of phytochrome-mediated responses to best suit the growth and development of the plant under the light conditions it encounters.

In contrast to our insight into the functions of phyA and phyB, much less is known about the function of other phytochromes. In this issue, Hennig et al. (pp. 194-200) report that phyE plays a role in controlling photo-induced seed germination in Arabidopsis. Previous studies have shown that both phyA and phyB mediate the photo-induction of seed germination by R light whereas the induction of seed germination by FR light is mediated only by phyA. However, a role for other phytochrome members in this process was indicated by the fact that phyA phyB double mutants still demonstrated R/FR-reversible induction of seed germination. Hennig et al. employed a set of photoreceptor mutants to test whether phyD or phyE or both can control photo-induced germination. Their results indicate that only phyB and phyE participate directly in R/FR reversible germination, but that phyE, unlike phyB, does not inhibit phyA-mediated germination. In fact, phyE is required for germination of Arabidopsis seeds in HIR conditions. This interaction of phyE with phyA, however, is not observed in other HIR responses, including the induction of cotyledon unfolding or agravitropic growth.

	A Papain Ortholog Expressed in Differentiating Xylem Elements

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Tonoplast rupture releases vacuolar contents into the cytoplasm of differentiating tracheary elements and is rapidly followed by cell death. Hydrolytic enzymes released during this process continue the post-mortem digestion of the cell. A Cys peptidase (XCP1) that is homologous to papain has previously been detected in Arabidopsis, and it is localized exclusively in the xylem. To determine whether XCP1 could be involved in tracheary element autolysis, Funk et al. (pp. 84-94) investigated the localization of XCP1 using XCP1 promoters fused to -glucuronidase and immunofluorescent confocal microscopy. Their results indicate that XCP1 is localized in the in the vacuole, consistent with it playing a role in tracheary element differentiation. The ectopic expression of XCP1 resulted in a range of phenotypes, with the most severely affected lines exhibiting stunting, increased anthocyanin levels, and early leaf senescence. The authors also present an intriguing hypothesis that the differentiation of laticifers may simply be a variation of the emerging model of tracheary element differentiation. They point out that differentiating tracheary elements and laticifers have many features in common, including their occurrence in the xylem, their accumulation of high levels of hydrolytic enzymes, and their formation of intercellular connections through end-wall perforations. Within the laticifer protoplast, however, only vesicles of ER origin are retained as the rest of the internal organelles, including the vacuole, become broken down. In laticifers, papain is localized in the ER vesicles, not in the central vacuole. Perhaps because of this, the complete autolysis of laticifers is prevented, and enzymes identical or paralogous to those used to catalyze the final steps of tracheary element autolysis are employed in laticifers as part of a pressurized defense network that is poised for the quick release of defensive peptidases.

	Systemic Induction of a Ca2+-Dependent Protein Kinase (CDPK)

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Plants undergo systemic physiological changes in response to local injuries caused by insects, pathogen attack, or mechanical wounding. The systemic wound-induced response is regulated by chemical factors including abscisic acid, jasmonic acid, oligosaccharides and the octadecapeptide systemin, and by physical signals such as hydraulic variation potentials and electrical activation potentials. An important step in the signal transduction pathways of many of these chemical and physical factors is a transient increase in cytoplasmic Ca²⁺ levels, and the activation of CDPKs. In this issue, Chico et al. (pp. 256-270) report upon their isolation of a cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) that encodes for a CDPK. LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors or H₂O₂. Moreover, a systemic increase in LeCDPK1 mRNA was detected upon wounding, and this was correlated with an increase in the activity of a soluble CDPK. These results suggest that the up-regulation of LeCDPK1 is an integral part of tomato's defense against both biotic and abiotic attacks.