Characterization of hybrid larch somatic embryo maturation by biochemical analyses and by a novel, fast mid-infrared approach.

The morphology of somatic embryos (SE) is not a sufficient criterion to determine the level of maturation and the optimal stage to transfer embryos for germination, unlike the biochemical components. This composition characterization in the laboratory is too restrictive to be considered at each maturation cycle, as would be necessary. It is, therefore, essential to consider alternative methods. The objectives of this work were to achieve a complete biochemical characterization of the embryos during their development, to serve as a reference and develop a characterization based on infrared spectrometry and chemometrics. During the precotyledonary stage (0-3 weeks of maturation), water content and glucose and fructose levels were high, which is consistent with SE development. After 4 weeks, the cotyledonary SE had a metabolism oriented towards the storage accumulation of lipids, proteins and starch, whereas raffinose only appeared from 8 weeks. Mid-infrared calibration models were developed for water, proteins, lipids, carbohydrates, glucose, fructose, inositols, raffinose, stachyose and starch contents with an r2 average of 0.84. A model was also developed to discriminate the weeks of SE maturation. Different classes of age were discriminated with at least 72% of accuracy. Infrared analysis of the SE based on their full biochemical spectral fingerprint revealed a very slight variation in composition between 7 and 9 weeks, information that is very difficult to obtain by conventional analysis methods. These results provide novel insights into the maturation of conifer SE and indicate that mid-infrared spectrometry could be an easy and effective method for SE characterization.

ABSCISIC ACID-DEFICIENT4 Has an Essential Function in Both cis-Violaxanthin and cis-Neoxanthin Synthesis.

Abscisic acid (ABA), a plant hormone synthesized from carotenoids, functions in seed germination and abiotic stress responses. ABA is derived from the cleavage of 9-cis-isomers of violaxanthin and neoxanthin, which are oxygenated carotenoids, also called xanthophylls. Although genes encoding enzymes responsible for most steps of the ABA biosynthesis pathway have been identified, enzymatic reactions leading to the production of these cis-isomers from trans-violaxanthin remain poorly understood. Two mutants that lack trans- and cis-neoxanthin, tomato (Solanum lycopersicum) neoxanthin-deficient1 (nxd1) and Arabidopsis (Arabidopsis thaliana) ABA-deficient4 (aba4), were identified previously, but only aba4 exhibited ABA-deficient phenotypes. No enzymatic activity was detected for ABA4 and NXD1 proteins, and their exact function remained unknown. To further investigate ABA4 and NXD1 function in Arabidopsis, we compared phenotypes of single and double mutants, and analyzed the effect of ABA4 overexpression on ABA and carotenoid accumulation in wild-type and mutant backgrounds. We provide convergent evidence that ABA4 is not only required for the formation of trans- and 9'-cis-neoxanthin from trans-violaxanthin, but also controls 9-cis-violaxanthin accumulation. While nxd1 produces high amounts of 9-cis-violaxanthin and ABA, aba4 nxd1 exhibits reduced levels in both leaves and seeds. Furthermore, ABA4 constitutive expression in nxd1 increases both 9-cis-violaxanthin and ABA accumulation. Subcellular localization of NXD1 protein in transient expression assays suggests that production of the NXD1-derived factor required for neoxanthin synthesis takes place in the cytosol. Finally, we postulate that ABA4, with additional unknown cofactor(s), is required for, or contributes to, trans-to-cis violaxanthin isomerase activity, producing both cis-xanthophyll precursors of ABA.