Light on perenniality: Para-dormancy is based on ABA-GA antagonism and endo-dormancy on the shutdown of GA biosynthesis.

Perennial para- and endo-dormancy are seasonally separate phenomena. Whereas para-dormancy is the suppression of axillary buds (AXBs) by a growing shoot, endo-dormancy is the short-day elicited arrest of terminal and AXBs. In hybrid aspen (Populus tremula x P. tremuloides) compromising the apex releases para-dormancy, whereas endo-dormancy requires chilling. ABA and GA are implicated in both phenomena. To untangle their roles, we blocked ABA biosynthesis with fluridone (FD), which significantly reduced ABA levels, downregulated GA-deactivation genes, upregulated the major GA3ox-biosynthetic genes, and initiated branching. Comprehensive GA-metabolite analyses suggested that FD treatment shifted GA production to the non-13-hydroxylation pathway, enhancing GA4 function. Applied ABA counteracted FD effects on GA metabolism and downregulated several GA3/4 -inducible α- and γ-clade 1,3-ß-glucanases that hydrolyze callose at plasmodesmata (PD), thereby enhancing PD-callose accumulation. Remarkably, ABA-deficient plants repressed GA4 biosynthesis and established endo-dormancy like controls but showed increased stress sensitivity. Repression of GA4 biosynthesis involved short-day induced DNA methylation events within the GA3ox2 promoter. In conclusion, the results cast new light on the roles of ABA and GA in dormancy cycling. In para-dormancy, PD-callose turnover is antagonized by ABA, whereas in short-day conditions, lack of GA4 biosynthesis promotes callose deposition that is structurally persistent throughout endo-dormancy.

Axillary buds are dwarfed shoots that tightly regulate GA pathway and GA-inducible 1,3-ß-glucanase genes during branching in hybrid aspen.

Axillary buds (AXBs) of hybrid aspen (Populus tremula×P. tremuloides) contain a developing dwarfed shoot that becomes para-dormant at the bud maturation point. Para-dormant AXBs can grow out after stem decapitation, while dormant AXBs pre-require long-term chilling to release them from dormancy. The latter is mediated by gibberellin (GA)-regulated 1,3-ß-glucanases, but it is unknown if GA is also important in the development, activation, and outgrowth of para-dormant AXBs. The present data show that para-dormant AXBs up-regulate GA receptor genes during their maturation, but curtail GA biosynthesis by down-regulating the rate-limiting GIBBERELLIN 3-OXIDASE2 (GA3ox2), which is characteristically expressed in the growing apex. However, decapitation significantly up-regulated GA3ox2 and GA4-responsive 1,3-ß-glucanases (GH17-family; α-clade). In contrast, decapitation down-regulated γ-clade 1,3-ß-glucanases, which were strongly up-regulated in maturing AXBs concomitant with lipid body accumulation. Overexpression of selected GH17 members in hybrid aspen resulted in characteristic branching patterns. The α-clade member induced an acropetal branching pattern, whereas the γ-clade member activated AXBs in recurrent flushes during transient cessation of apex proliferation. The results support a model in which curtailing the final step in GA biosynthesis dwarfs the embryonic shoot, while high levels of GA precursors and GA receptors keep AXBs poised for growth. GA signaling, induced by decapitation, reinvigorates symplasmic supply routes through GA-inducible 1,3-ß-glucanases that hydrolyze callose at sieve plates and plasmodesmata.

Optimization and kinetic characterization of recombinant 1,3-ß-glucanase production in Escherichia coli K-12 strain BL21/pETSD10 - a bioreactor scale study.

UNLABELLED: The Escherichia coli K-12 strain BL21/pETSD10 was used to produce recombinant endocellular 1,3-ß-glucanase. This enzyme is responsible for the hydrolysis of the glycosidic bond in specific polysaccharides with tracts of unsubstituted ß-1,3-linked glucosyl residues. Conditions for the overproduction were experimentally examined, and the optimal values of the process on a bioreactor scale were found by interpolation of the experimental data. Cell induction was preferred during log-phase with relatively high cell density at OD600 near 1·1 with 0·074 g l(-1) of Isopropyl ß-D-1-thiogalactopyranoside (IPTG). The higher concentration of IPTG favors high enzyme production but with an excess of ballast protein. 1,3-ß-glucanase production was favoured with moderate culture aeration (0·7-0·9 vvm) and moderate stirring (125-150 rev min(-1) ). The highest specific glucanase activity (252 U g(-1) ) was found during validated experiments carried out at aeration at 135 rev min(-1) and stirring at 0·8 vvm. Due to high-tonnage industrial applications (i.e. to hemicellulose hydrolysis), the enzymatic preparation did not need to be highly purified. After pretreatment (precipitation with ammonium sulphate and dialysis) of the crude preparation, the enzymatic protein was one of the three main proteins in the preparation. The reaction rate with respect to the substrate (CM-curdlan) was described by the first order reaction equation (k = 1·95 l h(-1 ) g(-1) ). Products formed in the reaction are composed of nine glucose units on average. In the reaction conditions, the preparation showed very good stability (t1/2 = 202 h). SIGNIFICANCE AND IMPACT OF THE STUDY: The results contribute to the knowledge of cultivation parameters of E. coli K-12 strain BL21/pETSD10 on a bioreactor scale to overproduce an enzyme degrading ß-1,3-glucans. The optimal values of protein concentration, specific activity and total glucanase activity as a function of aeration and stirring were evaluated by numerical analysis. The obtained values were validated as positive. The protein degrades some bonds in hemicellulose. Thus, the protein could be applied as one of the degrading components for hemicellulose.

Refurbishing the plasmodesmal chamber: a role for lipid bodies?

Lipid bodies (LBs) are universal constituents of both animal and plant cells. They are produced by specialized membrane domains at the tubular endoplasmic reticulum (ER), and consist of a core of neutral lipids and a surrounding monolayer of phospholipid with embedded amphipathic proteins. Although originally regarded as simple depots for lipids, they have recently emerged as organelles that interact with other cellular constituents, exchanging lipids, proteins and signaling molecules, and shuttling them between various intracellular destinations, including the plasmamembrane (PM). Recent data showed that in plants LBs can deliver a subset of 1,3-ß-glucanases to the plasmodesmal (PD) channel. We hypothesize that this may represent a more general mechanism, which complements the delivery of glycosylphosphatidylinositol (GPI)-anchored proteins to the PD exterior via the secretory pathway. We propose that LBs may contribute to the maintenance of the PD chamber and the delivery of regulatory molecules as well as proteins destined for transport to adjacent cells. In addition, we speculate that LBs deliver their cargo through interaction with membrane domains in the cytofacial side of the PM.