The tomato plastidic fructokinase SlFRK3 plays a role in xylem development.

Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.

LeFRK2 is required for phloem and xylem differentiation and the transport of both sugar and water.

It has been suggested that LeFRK2, the major fructose-phosphorylating enzyme in tomato plants, may be required for stem xylem development. Yet, we do not know if this enzyme affects the development of individual vessels, whether it affects water conductance, or whether it affects phloem development and sugar transport. Here, we show that suppression of LeFRK2 results in a significant reduction in the size of vascular cells and slows fiber maturation. The vessels in stems of LeFRK2-antisense plants are narrower than in WT plants and have thinner secondary cell walls. Although the cambium produces rounded secondary vessels, these vessels become deformed during the early stages of xylem maturation. Water conductance is then reduced in stems, roots, and leaves, suggesting that LeFRK2 influences xylem development throughout the entire vascular system. Interestingly, the build-up of positive xylem pressure under static (no-flow) conditions was also decreased. Suppression of LeFRK2 reduced the length and width of the sieve elements, as well as callose deposition. To examine the effect of LeFRK2 suppression on phloem transport, we created triple-grafted plants in which a portion of the wild-type stem was replaced with an antisense interstcok, and compared the contents of the transported sugar, sucrose, in the different portions of these stems. Sucrose contents above and within the LeFRK2-antisense interstock were significantly higher than those below the graft. These results show that the antisense interstock restricted the downward movement of sucrose, suggesting that LeFRK2 is required for both phloem and xylem development.

Spinach SoHXK1 is a mitochondria-associated hexokinase.

Hexokinase, a hexose-phosphorylating enzyme, has emerged as a central enzyme in sugar-sensing processes. A few HXK isozymes have been identified in various plant species. These isozymes have been classified into two major groups; plastidic (type A) isozymes located in the plastid stroma and those containing a membrane anchor domain (type B) located mainly adjacent to the mitochondria, but also found in the nucleus. Of all the hexokinases that have been characterized to date, the only exception to this rule is a spinach type B HXK (SoHXK1) that, by means of subcellular fractionation, has been localized to the outer membrane of plastids. However, SoHXK1 has a membrane anchor domain that is almost identical to that of the other type B HXKs. To determine the localization of SoHXK1 enzyme by other means, we expressed SoHXK1::GFP fusion protein in tobacco and Arabidopsis protoplasts and compared its localization with that of the Arabidopsis AtHXK1::GFP fusion protein that shares a similar N-terminal membrane anchor domain. SoHXK1::GFP is localized adjacent to the mitochondria, similar to AtHXK1::GFP and all other previously examined type B HXKs. Proteomic analysis had previously identified AtHXK1 on the outside of the mitochondrial membrane. We, therefore, suggest that SoHXK1 enzyme is located adjacent to the mitochondria like the other type B HXKs that share the same N-terminal membrane anchor domain.

Evidence for intracellular spatial separation of hexokinases and fructokinases in tomato plants.

Four hexokinase (LeHXK1-4) and four fructokinase (LeFRK1-4) genes were identified in tomato plants. Previous GFP fusion studies indicate that the gene product of LeHXK3 is associated with the mitochondria while that of LeHXK4 is located within plastids. In this study we found that the enzyme encoded by the fructokinase gene LeFRK3 is also located within plastids. The presence of LeFrk3 enzyme in plastids raises the question of the origin of fructose in these organelles. The other three FRKs enzymes, LeFrk1&2&4, are located in the cytosol. Unlike LeFrk1&2&4, the two additional HXKs, LeHxk1&2, share a common membrane anchor domain and are associated with the mitochondria similar to LeHxk3. The difference in the locations of the cytoplasmic FRK and HXK isozymes suggests that glucose phosphorylation is confined to defined special intracellular localizations while fructose phosphorylation is less confined.