On the Inside

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species, and it has been hypothesized that plants that load phloem symplastically have higher frequencies of plasmodesmata than do those that load phloem from the apoplast. A complication is that species that have high frequencies of plasmodesmata, and that have been presumed to be symplastic phloem loaders, fall into two categories dependinguponwhether they translocate raffinose-family oligosaccharides (RFOs) or sucrose. To date, the only detailed mechanism of symplastic loading that has been proposed—the polymer trap model— applies only to RFO species. This model is based on the correlation between the long-distance transport of RFOs and the presence of intermediary cells that abut bundle sheath cells (BSCs) and are linked to them by extremely numerous and asymmetrically branched plasmodesmata. According to the polymer trap model, Suc diffuses from BSCs into intermediary cells through the abundant plasmodesmata that connect the two cell types. In the intermediary cells, the Suc is converted to RFOs. Since RFOs are larger than Suc, they are unable to flow back into the BSCs through the plasmodesmata. Turgeon and Medville (pp. 3795–3803) reexamine the supposed correlation between high plasmodesmatal frequency and symplastic loading in three species that have abundant plasmodesmata in their minor veins. Their study indicates that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, Clethra barbinervis and Liquidambar styraciflua load from the apoplast. Thus, plasmodesmatal frequencies in theminor veins may have limited relevance to phloem loading. The authors suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast. Pollen-Papilla Interactions Affect Calcium Dynamics Although theCadynamics of growing pollen tubes have been well documented in vitro using germination assays and Ca imaging techniques, very few in vivo studies have been performed of the Ca dynamics in the pollen grain and papilla cell during pollination. Iwano et al. (pp. 3562– 3571) expressed yellow cameleon, one member of a new generation of fluorescent Ca indicators that are chimeric constructs of calmodulin and green fluorescent protein (GFP), in the pollen grains and papilla cells of Arabidopsis and monitored [Ca]cyt dynamics during pollination. In the pollen grain, [Ca]cyt increased at the potential germination site soon after hydration and remained at high levels until germination. Increased regions of [Ca]cyt in the tip of the elongating pollen tube were also observed (Fig. 1). Consistent with previous in vitro germination studies, [Ca]cyt oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca]cyt was higher than had been observed in vitro. This finding suggests that interactions with papillae may influence calcium dynamics within the pollen grain. The authors also report that the higher the frequency of these oscillations, the faster was the growth of the pollen tubes. In the pollinated papilla cell, dramatic 8-fold increases in [Ca]cyt occurred three times in succession, just under the site of pollen-grain attachment. [Ca]cyt increased first soon after pollen hydration, with a second increase occurring after pollenprotrusion. The third andmost remarkable [Ca]cyt increase took place when the pollen tube penetrated the papilla cell wall.

The incidence of plasmodesmata in the minor vein phloem of leaves varies widely between species, and it has been hypothesized that plants that load phloem symplastically have higher frequencies of plasmodesmata than do those that load phloem from the apoplast. A complication is that species that have high frequencies of plasmodesmata, and that have been presumed to be symplastic phloem loaders, fall into two categories depending upon whether they translocate raffinose-family oligosaccharides (RFOs) or sucrose. To date, the only detailed mechanism of symplastic loading that has been proposed-the polymer trap modelapplies only to RFO species. This model is based on the correlation between the long-distance transport of RFOs and the presence of intermediary cells that abut bundle sheath cells (BSCs) and are linked to them by extremely numerous and asymmetrically branched plasmodesmata. According to the polymer trap model, Suc diffuses from BSCs into intermediary cells through the abundant plasmodesmata that connect the two cell types. In the intermediary cells, the Suc is converted to RFOs. Since RFOs are larger than Suc, they are unable to flow back into the BSCs through the plasmodesmata. Turgeon and Medville (pp. 3795-3803) reexamine the supposed correlation between high plasmodesmatal frequency and symplastic loading in three species that have abundant plasmodesmata in their minor veins. Their study indicates that, contrary to the hypothesis that all species with abundant minor vein plasmodesmata load symplastically, Clethra barbinervis and Liquidambar styraciflua load from the apoplast. Thus, plasmodesmatal frequencies in the minor veins may have limited relevance to phloem loading. The authors suggest that symplastic loading is restricted to plants that translocate oligosaccharides larger than Suc, such as RFOs, and that other plants, no matter how many plasmodesmata they have in the minor vein phloem, load via the apoplast.

Pollen-Papilla Interactions Affect Calcium Dynamics
Although the Ca 21 dynamics of growing pollen tubes have been well documented in vitro using germination assays and Ca 21 imaging techniques, very few in vivo studies have been performed of the Ca 21 dynamics in the pollen grain and papilla cell during pollination.  expressed yellow cameleon, one member of a new generation of fluorescent Ca 21 indicators that are chimeric constructs of calmodulin and green fluorescent protein (GFP), in the pollen grains and papilla cells of Arabidopsis and monitored [Ca 21 ] cyt dynamics during pollination. In the pollen grain, [Ca 21 ] cyt increased at the potential germination site soon after hydration and remained at high levels until germination. Increased regions of [Ca 21 ] cyt in the tip of the elongating pollen tube were also observed ( Fig. 1). Consistent with previous in vitro germination studies, [Ca 21 ] cyt oscillations were observed in the tip region of the growing pollen tube, but the oscillation frequency was faster and [Ca 21 ] cyt was higher than had been observed in vitro. This finding suggests that interactions with papillae may influence calcium dynamics within the pollen grain. The authors also report that the higher the frequency of these oscillations, the faster was the growth of the pollen tubes. In the pollinated papilla cell, dramatic 8-fold increases in [Ca 21 ] cyt occurred three times in succession, just under the site of pollen-grain attachment. [Ca 21 ] cyt increased first soon after pollen hydration, with a second increase occurring after pollen protrusion. The third and most remarkable [Ca 21 ] cyt increase took place when the pollen tube penetrated the papilla cell wall.

Oxalate Mediates Fungal Infection by Affecting Stomatal Function
Several phytopathogenic fungi, including Sclerotinia sclerotiorum, produce millimolar concentrations of oxalate in infected tissues. Oxalate is an essential virulence factor of S. sclerotiorum be-cause mutants that are deficient in oxalate biosynthesis are less pathogenic than wild-type fungus. Moreover, enzymes that catabolize oxalate protect plants from Sclerotinia infection when their genes are expressed in stably transformed plants. The detailed mechanisms by which oxalic acid affects host cells and tissues, however, are not understood. In this issue, Guimarães and Stotz (pp. 3703-3711) examine the question of whether oxalate causes foliar dehydration following infection by disturbing guard cell function. The stomatal pores of Vicia faba leaves infected with S. sclerotiorum open at night. This cellular response appears to be dependent on oxalic acid, because stomatal pores are partially closed when leaves are infected with an oxalate-deficient mutant of S. sclerotiorum. The authors present evidence that the abnormal opening of stomatal pores may play a role in allowing S. sclerotiorum to emerge through open stomata from the uninfected abaxial leaf surface for secondary colonization. Consistent with a role for oxalate in this process, the exogenous application of oxalic acid to the detached abaxial epidermis of V. faba leaves induces stomatal opening, apparently by causing potassium accumulation and starch breakdown in the guard cells. Oxalate also interferes with abscisic acid (ABA)induced stomatal closure. A number of ABA-insensitive Arabidopsis mutants were found to be more susceptible to oxalate-deficient S. sclerotiorum than are wild-type plants, suggesting that Sclerotinia resistance is dependent on ABA. Thus, oxalate influences stomatal function by causing the accumulation of osmotically active molecules in guard cells and by inhibiting ABA-induced stomatal closure.

Cesium Toxicity in Arabidopsis
Although cesium (Cs) is chemically similar to potassium (K), K is an essential element, whereas Cs is toxic to plants. Two hypotheses have been put forth to explain Cs toxicity: (1) Extracellular Cs 1 prevents K 1 uptake, thereby inducing K starvation, and (2)  quotient. This is consistent with the hypothesis that Cs intoxication involves competition between K 1 and Cs 1 for K 1 -binding sites on essential proteins.

Calcium Dynamics During Nod Factor Signaling
The synthesis and perception of a variety of signaling molecules are required for the establishment of the symbiotic association between legumes and the soil bacteria rhizobia and the formation of nitrogen-fixing root nodules. Rhizobium-secreted Nod factors are key players in this molecular dialogue, eliciting developmental responses in both epidermal and the inner root tissues of the host plant that are essential for both bacterial entry and nodule organogenesis. Direct evidence for a role for Ca 21 in Nod factor signaling has come from measurements of rapid increases of intracellular Ca 21 and delayed regular Ca 21 spiking in response to Nod factors. Other pharmacological studies in transgenic alfalfa (Medicago trunculata) have suggested that Nod factor signal transduction in root hairs may also involve heterotrimeric G proteins that mediate downstream phosphoinositide-based signaling. In this issue,  show that a range of intracellular Ca 21 channel and pump antagonists previously shown to block Nod factor-elicited Ca 21 -spiking are also efficient inhibitors of the activation of a GUS reporter driven by an early nodulin gene promoter (pMtENOD12). They also provide evidence that multiple phospholipid signaling pathways, including phospholipases C and D, are likely to be central to the transduction of Nod factor perception at the root hair plasma membrane. Previous studies have revealed that the genes DMI1, 2, and 3 are essential for Rhizobium infection and nodulation and are involved in the earliest responses to Nod factor signaling in root hairs. Since dmi1 and dmi2 mutants (but not the dmi3 mutant) are defective in Nod factor-elicited Ca 21 spiking, it has been proposed that this characteristic calcium response is an integral element of the signaling pathway and positioned downstream of DMI1 and 2 and upstream of DMI3. The authors report that a mastoparan peptide agonist (Mas7) is able to elicit pMtENOD11-GUS transcription in all three dmi mutant backgrounds, suggesting that mastoparan acts at a step in the pathway between DMI1/DMI2 and DMI3.

Circadian Rhythms of Ethylene Emission
Previous studies have shown that ethylene emission and plant growth are under circadian regulation. The main control point for ethylene production in Arabidopsis is the synthesis of the precursor ACC. Thain et al. (pp. 3751-3761) show that the circadian clock in Arabidopsis drives the expression of multiple ACS (ACC SYNTHASE) genes, resulting in peak RNA levels at the phase of maximal ethylene synthesis. The rhythmic emanation of ethylene was particularly well correlated with ACS8 transcript levels. The expression of ACS8 is controlled by light, by the circadian clock, and by negative feedback regulation. In addition, ethylene production is controlled by the TOC1 (TIMING OF CAB EXPRESSION 1) and CCA1 (CIRCADIAN CLOCK-ASSOCIATED 1) genes, which appear to be critical for all circadian rhythms in Arabidopsis studied to date. Ethylene signaling mutants did not exhibit altered phases or periods of circadian ethylene emissions. In plants that lack pulvini, such as Arabidopsis, the circadian rhythm of leaf angle is driven by petiole elongation. Mutants with altered ethylene production or signaling also retained normal rhythmicity of leaf movement, indicating that circadian rhythms of ethylene production are not critical for rhythmic growth.

Peter V. Minorsky Department of Natural Sciences
Mercy College Dobbs Ferry, New York 10522