Role of ω-3 fatty acid desaturases in the regulation of the level of trienoic fatty acids during leaf cell maturation

Trienoic fatty acids, namely -linolenic acid and hexadecatrienoic acid, present in leaf lipids are produced by -3 fatty acid desaturases located in the endoplasmic reticulum and plastid membranes. The changes in the level of trienoic fatty acids during leaf maturation were investigated in wild-type plants of Arabidopsis thaliana (L.) Heynh. and in the fad7 mutant deficient in the activity of a plastid -3 desaturase. The levels of trienoic fatty acids increased in 26 °C- and 15 °C-grown wild-type plants with maturation of leaves. The increase in trienoic fatty acids was mainly due to galactolipids enriched in plastid membranes. In addition, the relative levels of trienoic fatty acids in major glycerolipids, including phospholipids enriched in the endoplasmic reticulum membranes, also increased with leaf maturation. By contrast, when the fad7 mutant was grown at 26 °C, the relative levels of trienoic fatty acids in individual lipids decreased with leaf maturation. The decreases in the levels of trienoic fatty acids, however, were alleviated when the fad7 mutant was grown at 15 °C. These results suggest that the plastid -3 desaturase plays a major role in increasing the levels of trienoic fatty acids with leaf maturation.Abbreviations 163 hexadecatrienoic acid - 183 -linolenic acid - DGD digalactosyldiacylglycerol - MGD monogalactosyldiacylglycerol - PC phosphatidylcholine - PE phosphatidylethanolamine - TA trienoic fatty acid - WT wild type - -3 refers to the position of the double bond from the methyl end of a fatty acid This research was supported in part by Grants-in-Aid for Scientific research (#07251214 and #06804050 to K.I.) from the Ministry of Education, Science and Culture, Japan, and by the research grant from Shorai Foundation.     

Impact of segmental chromosomal duplications on leaf size in the grandifolia-D mutants of Arabidopsis thaliana

The number of cells in an organ is a major factor that specifies its size. However, the genetic basis of cell number determination is not well understood. To obtain insight into this genetic basis, three grandifolia-D ( gra-D ) mutants of Arabidopsis thaliana were characterized that developed huge leaves with two to three times more cells than the wild-type. Genetic and microarray analyses showed that a large segmental duplication had occurred in all the gra-D mutants, consisting of the lower part of chromosome 4. In the duplications, genes were found that encode AINTEGUMENTA (ANT), a factor that extends the duration of cell proliferation, and CYCD3;1, a G₁/S cyclin. The expression levels of both genes increased and the duration of cell proliferation in the leaf primordia was extended in the gra-D mutants. Data obtained by RNAi-mediated knockdown of ANT expression suggested that ANT contributed to the huge-leaf phenotype, but that it was not the sole factor. Introduction of an extra genomic copy of CYCD3;1 into the wild-type partially mimicked the gra-D phenotype. Furthermore, combined elevated expression of ANT and CYCD3;1 enhanced cell proliferation in a cumulative fashion. These results indicate that the duration of cell proliferation in leaves is determined in part by the interaction of ANT and CYCD3;1 , and also demonstrate the potential usefulness of duplication mutants in the elucidation of genetic relationships that are difficult to uncover by standard single-gene mutations or gain-of-function analysis. We also discuss the potential effect of chromosomal duplication on evolution of organ size.     

Expression of a gene for plastid ω-3 fatty acid desaturase and changes in lipid and fatty acid compositions in light- and dark- grown wheat leaves

The leaves of monocotyledonous plants create a developmental sequence of cells and plastids from the base to the apical portion. We investigated fatty-acid and lipid compositions in successive leaf sections of light- and dark-grown wheat (Triticum aestivum L. cv. Chihoku) seedlings. The most notable change in the fatty acid composition was the increase of linolenic acid (18:3) with maturation of leaf cells, which occurred both in light- and dark-grown leaf tissues. In light-grown leaves, the increase of 18:3 with maturation was mainly attributed to the increase of monogalactosyldiacylglycerol (MGD) and also to the increase of the 18:3 level of MGD. In dark-grown leaves, the increase of 18:3 in the leaf apex was caused by the increase of the levels of MGD and digalactosyldiacylglycerol (DGD) and also by the increase of the 18:3 levels of within these two lipids. Since MGD and DGD are mainly found in plastid membranes, these findings indicate that both the synthesis of galactolipids and the formation of 18:3 these lipids take place during plastid development. The plastid ω-3 fatty acid desaturase is responsible for the formation of 18:3 in plastid membrane lipids. To investigate the regulation of desaturation, we isolated a gene for wheat plastid ω-3 fatty acid desaturase (TaFAD7). The mRNA level of TaFAD7 in light-grown leaves was much higher than that in dark-grown leaves. During the greening of etiolated leaves the level of TaFAD7 mRNA increased significantly, accompanied by an increase of the 18:3 level of total fatty acids. On the other hand, the levels of TaFAD7 mRNA were almost the same in all the leaf sections of both light- and dark-grown leaf tissues. These results suggest that the effect of the expression of the TaFAD7 gene on the increase of the 18:3 level is different between the leaf development under continuous light- or dark-conditions and the light-induced greening process of etiolated leaves. The increase of 18:3 content of MGD (or MGD and DGD) with maturation is apparently regulated not solely by the level of TaFAD7 mRNA.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

Genetic Relationship Between Angustifolia3 and Extra-Small Sisters Highlights Novel Mechanisms Controlling Leaf Size

Coordination between cell proliferation and cell expansion is pivotal in leaf size determination. A group of mutants that are impaired in cell proliferation such as the angustifolia3 (an3) has provided a clue to understanding how these cellular processes are coordinated. In these mutants, impaired cell proliferation is accompanied by enhanced cell enlargement. We propose to call this phenomenon “compensated cell enlargement.” Previously, we isolated ten extra-small sisters (xs) mutants that are specifically impaired in post-mitotic cell expansion and found that several xs mutations are able to suppress compensated cell enlargement in an3. Thus, the enhanced cell expansion observed in an3 results from the hyperactivation of post-mitotic cell expansion involving specific members of the XS gene family. These results suggested that cell proliferation process(es) and post-mitotic cell expansion process(es) are somehow linked in an as yet unknown fashion in leaf primordia. In this addendum, we propose possible models for the linking mechanisms that coordinate AN3-dependent cell proliferation and XS-dependent cell expansion in leaf development.Key Words: Arabidopsis, cell expansion, cell proliferation, extra-small sisters (xs), angustifolia3 (an3), compensated cell enlargement, leaf, organ size controlMature leaf size is determined by the final number and size of cells within a leaf. Thus, the spatial and temporal regulation of cell proliferation and cell expansion plays pivotal roles in establishing developmentally programmed leaf size in a reproducible fashion. During leaf development, cell proliferation is maintained in the basal part of the leaf primordium and terminates basipetally.^1,2 Cells that exit cell cycling undergo differentiation and expand enormously.¹ Although many studies have revealed the molecular mechanisms underlying the above processes, the coordination of cell proliferation and cell expansion in the context of organogenesis is not yet understood.In recent years, an interesting phenomenon has been reported in leaves of several mutants or transgenic plants with impaired cell proliferation. These mutants not only have a defect in cell proliferation, but have larger cells than does the wild type, suggesting that the cell proliferation process interacts with the cell expansion process during leaf organogenesis.^3–8 This phenomenon, called “compensation,” has highlighted the existence of a coordination system between cell proliferation and cell expansion in leaf development.^9–13Conceptually, this compensation can be dissected into two processes: the induction process involves the reduction of cell proliferation and the response process directs the enhancement of cell expansion and “compensated cell enlargement.” Recently, we showed that, in the typical compensation-exhibiting mutant angustifolia3 (an3), the expansion of post-mitotic, but not mitotic, cells is specifically enhanced.⁸ Thus, the induction and the response processes should take place separately in proliferating and differentiating cells, respectively. To dissect compensation genetically, with an emphasis on the response process, we isolated 10 mutants, named extra-small sisters (xs), that are specifically impaired in post-mitotic cell expansion.^14–16 We classified xs mutants into three classes based on the effect of each xs mutation on compensated cell enlargement, using an3 as a representative of compensation-exhibiting mutants.¹⁴ As expected, a group of xs mutants (xs1, xs2, xs4 and xs5) completely suppressed compensated cell enlargement in an3 mutants (named the “small-cell” class), whereas the other two classes had either no suppressive or additive effects on cell enlargement.¹⁴ This finding demonstrated that these XS genes act downstream of cell expansion pathways that are regulated by compensation and triggered in an3. How is this relationship established in the context of leaf organogenesis? When considering the above result, one might speculate that, in addition to a promotive role in cell proliferation, AN3 has a role in cell expansion post-mitotic cells. However, AN3 is hardly expressed in differentiating cells, and the overexpression of AN3 has no effects on post-mitotic cell expansion,⁶ suggesting that this possibility is unlikely. Taken together, these results indicate that the AN3-dependent cell proliferation pathway is somehow linked by an intermediary process to post-mitotic cell expansion pathway(s) involving the small-cell class XS.Based on these data, we propose two possible scenarios for the intermediary process, categorized in terms of cell autonomy (Fig. 1). In the non-cell-autonomous case, proliferating cells located in the basal region of the developing leaf may regulate the expansion of differentiating cells located in the upper region of the leaf via unknown cell-cell communications (Fig. 1A and B). In the cell-autonomous case, the activity involved in cell proliferation may be memorized in each cell, and, depending on this memory, each post-mitotic cell determines its own final size (Fig. 1C). Whatever the mechanism, we can assume that regulatory signal(s) would be affected by cell proliferation. Irrespective of cell autonomy, this putative signal acts either positively or negatively on cell expansion. When cell proliferation is impaired by the an3 mutation, the strength of the negative signal would be reduced and become insufficient to prevent differentiating cells from excessive cell expansion. Conversely, if this signal plays a positive role in cell expansion, the signal may be insufficient to positively control cell expansion in the wild type. However, when the cell number is significantly reduced by the an3 mutation, this positive signal(s) would hyperactivate cell expansion pathways. Further analyses of the factors involved in the intermediary process should provide an important insight into signaling mechanisms that control leaf size.Open in a separate windowFigure 1Proposed models for the leaf size control inferred from the analysis of compensated cell enlargement. (A and B) Non-cell-autonomous model. Cells located in the basal part of a leaf primordium would produce signal(s) that inhibit (A) or promote (B) cell expansion of post-mitotic cells present in the apical part of the leaf primordium. (A) If a significant reduction in cell number occurs in an3, the strength of the inhibitory signal would be reduced and cell expansion would be de-repressed, resulting in the abnormal enlargement of leaf cells. (B) When we assume a cell expansion-promoting signal(s), its strength may be insufficient to enhance cell expansion in the wild type. When a significant reduction in cell number occurs in an3, the promoting signal(s) would increase sufficiently to cause compensation. (C) “Cell memory” model. A specific signal reflecting cell proliferation activity in proliferating cells is retained during cell differentiation and affects the magnitude of cell expansion. Inhibitory and stimulatory signal examples are shown in the upper and lower panels, respectively. The relationship between the strength of the signals and the induction of compensation is the same as that described in the non-cell-autonomous model.Before our reports on the actions of an3, fugu and xs mutants,^8,14 compensated cell enlargement was considered to be caused by the uncoupling of cell division and growth. Now, this possibility is clearly ruled out. Cell proliferation and post-mitotic cell expansion are the most basic cellular processes and are each supported by different regulatory networks. The putative signaling systems discussed here provide a new perspective on how developmental programs integrate these networks into a super-network to control organ size.