Transgenic Poplar Plants for the Investigation of ABA-Dependent Salt and Drought Stress Adaptation in Trees

Important functions of the plant hormone abscisic acid (ABA) in stress reactions, growth and photosynthetic processes are extensively studied in the model plant Arabidopsis thaliana. This paper investigates the importance of Moco-sulphurase ABA3 and aldehyde oxidase (AO) on ABA-biosynthesis in Populus × canescens. ABA3 is essential for activation of the molybdenum enzymes AO and xanthine dehydrogenase (XDH). AO itself catalyzes the last step in ABA-biosynthesis. Generation of transgenic poplar plants altered in ABA3 and AO-activity using RNAi knock down and overexpression was performed. Whereas RNAi-AO plants show a specific loss of AO activity, the RNAi-ABA3 plants has a strongly reduced activity of both molybdenum enzymes: AO and XDH. Constructs of AO and ABA3-promoters fused to β-glucuronidase provide the basis to investigate transcriptional regulation of ABA-biosynthetic processes under stress conditions. Application of high salt concentrations and different drought stress intensities does change the endogenous AO or XDH neither on the side of transcription nor on protein activity. On phytohormone level however, water loss leads to increased ABA-amounts regardless of whether transgenic or wildtype plants are studied. Salt application resulted in higher ABA-levels in all analyzed plant lines. The down regulation of AO in the two different RNAi-plant lines strongly prevented a wildtype-like increase of ABA-levels. Whereas the WT plants accumulated up to 6000 ng ABA g-1 FW-1 after 16 h of salt stress exposure, plants of the RNAi lines revealed a markedly lower increase of only up to 2000 ng ABA g-1 FW-1. Opposing to these observations, ABA-levels increased during drought without any influence by the RNAi-effect. These results revealed that although stresses did not result in a visible increased AO-activity, ABA-production was influenced by AO and ABA3 at least under salinity.

An alternative,zeaxanthin epoxidase-independent abscisic acid biosynthetic pathway in plants

Abscisic acid(ABA)is an important carotenoid-derived phytohormone that plays essential roles in plant response to biotic and abiotic stresses as well as in various physiological and developmental processes.In Arabidopsis,ABA biosynthesis starts with the epoxidation of zeaxanthin by the ABA DEFICIENT 1(ABA1)enzyme,leading to epoxycarotenoids;e.g.,violaxanthin.The oxidative cleavage of 9-cis-epoxycaro-tenoids,a key regulatory step catalyzed by 9-C/S-EPOXYCAROTENOID DIOXYGENASE,forms xanthoxin,which is converted in further rea.ctions mediated by ABA DEFICIENT 2(ABA2),ABA DEFICIENT 3(ABA3),and ABSCISIC ALDEHYDE OXIDASE 3(AAO3)into ABA.By combining genetic and biochemical approaches,we unravel here an ABA1-independent ABA biosynthetic pathway starting upstream of zeaxanthin.We iden-tified the carotenoid cleavage products(i.e.,apocarotenoids,β-apo-11-carotenal,9-cis-β-apo-11-carotenal,3-OH-β-apo-11-carotenal,and 9-cis-3-OH-β-apo-11-carotenal)as intermediates of this ABA1-independent ABA biosynthetic pathway.Using labeled compounds,we showed thatβ-apo-11-carotenal,9-cis-β-apo-11-carotenal,and 3-OH-β-apo-11-carotenal are successively converted into 9-cis-3-OH-β-apo-11-carotenal,xanthoxin,and finally into ABA in both Arabidopsis and rice.When applied to Arabidopsis,theseβ-apo-11-carotenoids exert ABA biological functions,such as maintaining seed dormancy and inducing the expression of ABA-responsive genes.Moreover,the transcdptomic analysis revealed a high overlap of differentially expressed genes regulated byβ-apo-11-carotenoids and ABA,suggesting thatβ-apo-11-carot-enoids exert ABA-independent regulatory activities.Taken together,our study identifies a biological function for the common plant metabolites,β-apo-11-carotenoids,extends our knowledge about ABA biosynthesis,and provides new insights into plant apocarotenoid metabolic networks.