No abstract

Although the enucleate conducting cells of the phloem are incapable of protein synthesis, phloem exudates characteristically contain low concentrations of soluble proteins. The role of these proteins and their movement into and out of the sieve tubes poses important questions for phloem physiology and for cell-to-cell protein movement via plasmodesmata. Because mature sieve elements lack both a nucleus and ribosomes (4), they are incapable of protein synthesis. Clearly, the ongoing presence of proteins in the translocation stream requires their continual replacement by movement from adjacent nucleate cells. Companion cells are the most likely origin of such proteins and characteristically possess an active-appearing cytoplasm, including abundant ribosomes. Recently, Nakamura et al. (16) (17) showed that even 'structural' P-proteins are involved in rapid turnover and presented microautoradiographic evidence for their synthesis in companion cells.The occurrence of protein turnover in sieve tubes raises a number of intriguing and physiologically significant questions. Movement of proteins into and out of the sieve tube presumably occurs via plasmodesmata, which, except for pathological conditions, appear to provide passageways too small for intercellular protein movement (21). Although their functions are unknown, the proteins presumably have some role in source-sink and/or sieve tube-companion cell relations. As Raven (20) recently emphasized, the striking longevity of sieve elements as enucleate cells poses ongoing maintenance problems that almost certainly require intercellular protein transport. Finally, the synthesis and movement of phloem proteins in healthy plants may provide insight into the replication and movement of phloem-limited viruses and Mycoplasma-like organisms.The following experiments were undertaken to investigate some of the overall characteristics of soluble sieve tube proteins, especially their number and variability along the transport pathway, and their pattems of synthesis, transport, and turnover. MATERIALS AND METHODS Plant MaterialWheat plants (Triticum aestivum L. cv SUN 9E) were grown in a growth chamber as described previously (9). Experiments were performed with plants in the middle portion of the grain-filling stage (approximately 15-25 d after anthesis).

SummaryConfocal laser scanning microecopy (CLSM) has been used to Image phloem transport and unloading in the root tip of Atwbidopmis. The fluorescent probe 5(6) carboxyfluorascein (CF) was ester loaded into a single cotyledon end the entire seedling placed within an observation chamber under the micrcmcope. Translocation of CF to the root tip was rapid, followed by unloading into discrete concentric flies of ceils. The position of the prominent unloading 'zone' corresponded precilmly with that of the two protophloem files of sieve elements, demonstrating s functional role of these cells in symplsatic sieve-element unloading. Symplsatic transport following unloading was confined to the elongating zone of the root with little besipetal transport to more mature cells. Following photobieaching of the unloading zone, phloem transport was restored immediately into the protophloem sieve elements, followed rapidly by lateral, symplastic sieve-element unloading. The results demonstrate that phloem transport processes can now be Imaged in real time, and non-invasively, within an intact plant system.

All flowering plants produce S-methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that l-SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( approximately 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, l-SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given l-(35)S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis (S-adenosylMet:Met S-methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora, maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( approximately 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5'-phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase.

An analysis of the entrance and discharge of the pollen tube into the embryo sac of Gossypium hirsutum was made with the light and electron microscopes. The following sequence of events is seen in cotton: 1. One of the two synergids begins to degenerate following pollination but before the pollen tube reaches the embryo sac. This degeneration is marked by the swelling and darkening of the organelle membranes, the collapse of the vacuoles, and the disappearance of the plasma membrane. Striking chemical changes accompany the structural degeneration. 2. The pollen tube grows into the degenerating synergid through the filiform apparatus. The tube ceases growth while the tip is still in the synergid. A pore develops on the chalazal side of the tube in a subterminal position. 3. The pollen tube cytoplasm and the sperm are discharged into the degenerating synergid through the pore in the tube. Following discharge a plug forms at the pore. None of the discharge leaves the synergid except the sperm nuclei which enter the egg or central cell directly from the synergid. The X-bodies present in the synergid are the remains of the sperm cytoplasm. The data stress the importance of the degenerating synergid in pollen tube discharge and the entrance of the sperm nuclei into the egg and central cell. A hypothesis is presented to explain the passage of the sperm nuclei into the egg and central cell. The data show clearly that the pollen tube does not destroy the synergid it enters, and that the degenerating synergid following pollen-tube discharge is remarkably stable.

The use of exuding stylets holds considerable promise for the investigation of sieve-tube physiology. However, largely because of difficulties in cutting insect stylets, the technique has been applied to only a few plant species. Based on our experience, a comparison is made of the available means of obtaining sieve-tube exudate from the exuding stylets of phloem-feeding insects, including aphids, scale and mealybugs. Forty-one plant species and approx. 35 insect species were tested for their ability to provide stylet exudate. Stylets on all but a few of the plant species tested yielded at least some exudate, but the success rate and duration of exudation on many species were unsatisfactory for detailed investigations of phloem transport. Plant species appears to be the most important factor for obtaining reliably exuding stylets, although the size of the insect species used and the physiological condition of the plant are also important variables. Armored scale provide a simple and reliable source of exuding stylets, but are impractical for most experimental purposes. Radio-frequency microcautery of aphid stylets was substantially the most effective means of cutting stylets. Instructions are provided for constructing a microcautery unit at minimal expense, using a citizen's band radio as the radio-frequency source.

All flowering plants produce S -methylmethionine (SMM) from Met and have a separate mechanism to convert SMM back to Met. The functions of SMM and the reasons for its interconversion with Met are not known. In this study, by using the aphid stylet collection method together with mass spectral and radiolabeling analyses, we established that L -SMM is a major constituent of the phloem sap moving to wheat ears. The SMM level in the phloem ( ‫ف‬ 2% of free amino acids) was 1.5-fold that of glutathione, indicating that SMM could contribute approximately half the sulfur needed for grain protein synthesis. Similarly, L -SMM was a prominently labeled product in phloem exudates obtained by EDTA treatment of detached leaves from plants of the Poaceae, Fabaceae, Asteraceae, Brassicaceae, and Cucurbitaceae that were given L -35 S-Met. cDNA clones for the enzyme that catalyzes SMM synthesis ( S -adenosylMet:Met S -methyltransferase; EC 2.1.1.12) were isolated from Wollastonia biflora , maize, and Arabidopsis. The deduced amino acid sequences revealed the expected methyltransferase domain ( ‫ف‬ 300 residues at the N terminus), plus an 800-residue C-terminal region sharing significant similarity with aminotransferases and other pyridoxal 5 -phosphate-dependent enzymes. These results indicate that SMM has a previously unrecognized but often major role in sulfur transport in flowering plants and that evolution of SMM synthesis in this group involved a gene fusion event. The resulting bipartite enzyme is unlike any other known methyltransferase. INTRODUCTIONPlant Met metabolism differs from that in other organisms by involving S -methylmethionine (SMM). SMM is a ubiquitous constituent of the free amino acid pool in flowering plants, occurring in leaves, roots, and other organs at levels that typically range from 0.5 to 3 mol g Ϫ 1 dry weight, a concentration that is often higher than those of Met or S -adenosylmethionine (AdoMet) (Giovanelli et al., 1980;Mudd and Datko, 1990;Bezzubov and Gessler, 1992). SMM also has been detected as a metabolite of radiolabeled L -Met in all flowering plants tested ( Ͼ 50 species from Ͼ 20 families; Paquet et al., 1995). As shown in Figure 1, SMM is formed from L -Met via the action of AdoMet:Met S -methyltransferase (MMT; EC 2.1.1.12) and can be reconverted to Met by donating a methyl group to L -homocysteine (Hcy) in a reaction catalyzed by Hcy S -methyltransferase (HMT; EC 2.1.1.10; Giovanelli et al., 1980;Mudd and Datko, 1990). The tandem action of MMT and HMT, together with S -adenosyl-L -Hcy hydrolase, constitutes the SMM cycle, which is apparently futile (Mudd and Datko, 1990).As expected from the universality of SMM, MMT activity has been found in many flowering plants (Giovanelli et al., 1980;Mudd and Datko, 1990). It has been purified from leaves of Wollastonia biflora (James et al., 1995a) and from germinating barley (Pimenta et al., 1998), and it is known to have subunits of ‫ف‬ 115 kD. Because this is approximately three times larger than any other small-molecule methyltransferase (F...

All available evidence suggests that C4 plants have evolved from ancestors possessing the C3 pathway of photosynthesis and this has occurred independently many times in taxonomically diverse groups (3,21). At present, the precise evolutionary transition, at the anatomical, physiological, and biochemical levels, from a C3 to a C4 plant is not clear. It is generally believed that studies of C3-C4 intermediate species might provide insight into the evolution of C4 photosynthesis. In addition, since most of the world's important crops are C3 plants, there has been considerable interest in improving their productivity by screening for mutants with reduced rates of photorespiration or by incorporating C4 characteristics into C3 plants (3,19,20). Thus, the search for naturally '