Regulation of fructan metabolism in barley leaves

Abstract

Fructans, the polymers of fructose (Fru), are major non-structural storage carbohydrates in the vegetative tissues of many higher plants including temperate forage grasses and cereals, as well as major crop plants such as wheat and barley. Fructans play an important role in assimilate partitioning, plant development, environmental stress tolerance etc. Fructans also have a vast application potential in nutrition and medicine. The main focus of this dissertation is the fructan biosynthetic pathway in barley leaves. Its major aspects are the identification of sucrose:sucrose 1-fructosyltransferase (1-SST) as a pacemaker enzyme, regulation of the promoter of sucrose:fructan 6-fructosyltransferase (6-SFT) - one of the main fructosyltransferases (FTs) and the role of vacuolar invertases during fructan metabolism. Excised barley leaves exposed to continuous light accumulate large amounts of fructans containing β(2-6) linkages with β(2-1) branches, the so-called graminans. The pathway for graminan biosynthesis has not been well characterised, but it has been proposed that the successive action of two main enzymes, 1-SST and 6-SFT is involved (1-SST/6-SFT model). To demonstrate the validity of this model, excised leaves were subjected to a light-dark regime known to sequentially induce fructan accumulation and mobilization. The pattern of accumulation of soluble carbohydrates, the level of 1-SST and 6-SFT activities, and the expression of the corresponding genes, all indicate that the diversion of sucrose (Suc) into the pathway fructan synthesis is initiated by 1-SST induction. The stability of transcripts and enzyme activities of 1-SST and 6-SFT were compared, using appropriate inhibitors. The transcripts of 1-SST and enzymatic activity are subject to a rapid turnover and respond more quickly than 6-SFT. The much higher responsiveness of 1-SST to regulatory processes clearly indicates that it plays the role of the pacemaker enzyme of fructan synthesis in barley leaves. Plants regulate fructan synthesis in response to several internal and external stimuli primarily through the modulation of gene expression of FTs. Little is known about signal perception and transduction events that control the expression of FT genes. The regulatory sequences of FT genes are valuable tools to decipher the underlying signaling events. Using PCR-based genome walking procedures, the promoter of 6-SFT gene corresponding to 1.6 kb of the upstream region of the coding sequence, was cloned. The promoter activity of the cloned sequence was investigated in transient assays by fusing it to a reporter gene [uidA encoding β-glucuronidase, (GUS)] and by microprojectile bombardment of excised barley leaves. Strong expression of the GUS gene was observed in leaves induced for fructan biosynthesis by Suc and light, indicating that the cloned sequence contains the necessary cis acting elements conferring Suc and light induction of 6-SFT transcription. Arabidopsis thaliana has been extensively used to study the sugar induced signal transduction pathways in plants. In order to investigate the signaling events involved in the activation of the 6-SFT promoter, stably transformed Arabidopsis plants harboring the 6-SFT promoter driving the expression of the GUS reporter gene, were obtained. Though Arabidopsis is a non-fructan producing plant, the sugar-regulated activation of the barley 6-SFT promoter is maintained in Arabidopsis. The inhibitors of protein phosphatases and protein kinases, and a chelator of calcium, known to block Suc induction of 6-SFT gene expression in wheat, were effective in Arabidopsis too, suggesting that this signal transmission process seems to be conserved between cereals and Arabidopsis. These transgenic plants are valuable to study the activity of the barley 6-SFT promoter further and identify the transcription factors that interact with the key promoter elements. Invertases play a central role in the metabolism of Suc, the main product of photosynthesis and substrate for the synthesis of the fructans. Soluble acid invertase (SAI) isoforms are present in the vacuoles and are believed to be the ancestors of fructosyltransferases FTs. No SAI sequences are available from barley yet. In the present work, a soluble acid invertase cDNA was cloned from barley (HvSAI) and functionally characterized by heterologous expression in Pichia pastoris. Furthermore, the expression of HvSAI gene was studied in excised leaves and roots. The recombinant HvSAI cleaves Suc efficiently, but despite very high amino acid sequence similarity to FTs, is devoid of FT or fructan hydrolase like side activities. Compared to the FTs, the activity of the recombinant HvSAI is relatively easily saturable (Km of 13.5 mM for Suc) and possesses a higher temperature optimum (10°C more that 1-SST). The mRNA levels of HvSAI are constitutive and not affected much by enhanced sugar levels in excised leaves and roots, by Suc supply or continuous illumination of cut leaves. The cloning of SAIs will help to investigate their role in the regulation of fructan metabolism and decipher the structure-function relationship between SAI and FTs.