The N-terminal Part of Arabidopsis thaliana Starch Synthase 4 Determines the Localization and Activity of the Enzyme.

Starch synthase 4 (SS4) plays a specific role in starch synthesis because it controls the number of starch granules synthesized in the chloroplast and is involved in the initiation of the starch granule. We showed previously that SS4 interacts with fibrillins 1 and is associated with plastoglobules, suborganelle compartments physically attached to the thylakoid membrane in chloroplasts. Both SS4 localization and its interaction with fibrillins 1 were mediated by the N-terminal part of SS4. Here we show that the coiled-coil region within the N-terminal portion of SS4 is involved in both processes. Elimination of this region prevents SS4 from binding to fibrillins 1 and alters SS4 localization in the chloroplast. We also show that SS4 forms dimers, which depends on a region located between the coiled-coil region and the glycosyltransferase domain of SS4. This region is highly conserved between all SS4 enzymes sequenced to date. We show that the dimerization seems to be necessary for the activity of the enzyme. Both dimerization and the functionality of the coiled-coil region are conserved among SS4 proteins from phylogenetically distant species, such as Arabidopsis and Brachypodium This finding suggests that the mechanism of action of SS4 is conserved among different plant species.

Starch synthase 4 is located in the thylakoid membrane and interacts with plastoglobule-associated proteins in Arabidopsis.

Starch synthesis requires the formation of a primer that can be subsequently elongated and branched. How this primer is produced, however, remains unknown. The control of the number of starch granules produced per chloroplast is also a matter of debate. We previously showed starch synthase 4 (SS4) to be involved in both processes, although the mechanisms involved are yet to be fully characterised. The present work shows that SS4 displays a specific localization different from other starch synthases. Thus, this protein is located in specific areas of the thylakoid membrane and interacts with the proteins fibrillin 1a (FBN1a) and 1b (FBN1b), which are mainly located in plastoglobules. SS4 would seem to be associated with plastoglobules attached to the thylakoids (or to that portion of the thylakoids where plastoglobules have originated), forming a complex that includes the FBN1s and other as-yet unidentified proteins. The present results also indicate that the localization pattern of SS4, and its interactions with the FBN1 proteins, are mediated through its N-terminal region, which contains two long coiled-coil motifs. The localization of SS4 in specific areas of the thylakoid membrane suggests that starch granules are originated at specific regions of the chloroplast.

Arabidopsis thaliana plastoglobule-associated fibrillin 1a interacts with fibrillin 1b in vivo.

Plant fibrillins are a well-conserved protein family found in the plastids of all photosynthetic organisms, where they perform a wide range of functions. A number of these proteins have been suggested to be involved in the maintenance of thylakoids and the formation of plastoglobules, preventing their coalescence and favoring their clustering via an as-yet unidentified cross-linking mechanism. In this work we show that two members of this group, namely fibrillin 1a and 1b, interact with each other via a head-to-tail mechanism, thus raising the possibility that they form homo- or hetero-oligomers and providing a mechanism to understand the function of these proteins.

Enhancing the expression of starch synthase class IV results in increased levels of both transitory and long-term storage starch.

Starch is an important renewable raw material with an increasing number of applications. Several attempts have been made to obtain plants that produce modified versions of starch or higher starch yield. Most of the approaches designed to increase the levels of starch have focused on the increment of the amount of ADP-glucose or ATP available for starch biosynthesis. In this work, we show that the overexpression of starch synthase class IV (SSIV) increases the levels of starch accumulated in the leaves of Arabidopsis by 30%-40%. In addition, SSIV-overexpressing lines display a higher rate of growth. The increase in starch content as a consequence of enhanced SSIV expression is also observed in long-term storage starch organs such as potato tubers. Overexpression of SSIV in potato leads to increased tuber starch content on a dry weight basis and to increased yield of starch production in terms of tons of starch/hectare. These results identify SSIV as one of the regulatory steps involved in the control of the amount of starch accumulated in plastids.

Starch granule initiation in Arabidopsis requires the presence of either class IV or class III starch synthases.

The mechanisms underlying starch granule initiation remain unknown. We have recently reported that mutation of soluble starch synthase IV (SSIV) in Arabidopsis thaliana results in restriction of the number of starch granules to a single, large, particle per plastid, thereby defining an important component of the starch priming machinery. In this work, we provide further evidence for the function of SSIV in the priming process of starch granule formation and show that SSIV is necessary and sufficient to establish the correct number of starch granules observed in wild-type chloroplasts. The role of SSIV in granule seeding can be replaced, in part, by the phylogenetically related SSIII. Indeed, the simultaneous elimination of both proteins prevents Arabidopsis from synthesizing starch, thus demonstrating that other starch synthases cannot support starch synthesis despite remaining enzymatically active. Herein, we describe the substrate specificity and kinetic properties of SSIV and its subchloroplastic localization in specific regions associated with the edges of starch granules. The data presented in this work point to a complex mechanism for starch granule formation and to the different abilities of SSIV and SSIII to support this process in Arabidopsis leaves.

Metabolic and transcriptomic adaptation of Lactococcus lactis subsp. lactis Biovar diacetylactis in response to autoacidification and temperature downshift in skim milk.

For the first time, a combined genome-wide transcriptome and metabolic analysis was performed with a dairy Lactococcus lactis subsp. lactis biovar diacetylactis strain under dynamic conditions similar to the conditions encountered during the cheese-making process. A culture was grown in skim milk in an anaerobic environment without pH regulation and with a controlled temperature downshift. Fermentation kinetics, as well as central metabolism enzyme activities, were determined throughout the culture. Based on the enzymatic analysis, a type of glycolytic control was postulated, which was shared by most of the enzymes during the growth phase; in particular, the phosphofructokinase and some enzymes of the phosphoglycerate pathway during the postacidification phase were implicated. These conclusions were reinforced by whole-genome transcriptomic data. First, limited enzyme activities relative to the carbon flux were measured for most of the glycolytic enzymes; second, transcripts and enzyme activities exhibited similar changes during the culture; and third, genes involved in alternative metabolic pathways derived from some glycolytic metabolites were induced just upstream of the postulated glycolytic bottlenecks, as a consequence of accumulation of these metabolites. Other transcriptional responses to autoacidification and a decrease in temperature were induced at the end of the growth phase and were partially maintained during the stationary phase. If specific responses to acid and cold stresses were identified, this exhaustive analysis also enabled induction of unexpected pathways to be shown.