Crocus genome reveals the evolutionary origin of crocin biosynthesis

Crocus sativus (saffron) is a globally autumn-flowering plant, and its stigmas are the most expensive spice and valuable herb medicine. Crocus specialized metabolites, crocins, are biosynthesized in distant species, Gardenia (eudicot) and Crocus (monocot), and the evolution of crocin biosynthesis remains poorly understood. With the chromosome-level Crocus genome assembly, we revealed that two rounds of lineage-specific whole genome triplication occurred, contributing important roles in the production of carotenoids and apocarotenoids. According to the kingdom-wide identification, phylogenetic analysis, and functional assays of carotenoid cleavage dioxygenases (CCDs), we deduced that the duplication, site positive selection, and neofunctionalization of Crocus-specific CCD2 from CCD1 members are responsible for the crocin biosynthesis. In addition, site mutation of CsCCD2 revealed the key amino acids, including I143, L146, R161, E181, T259, and S292 related to the catalytic activity of zeaxanthin cleavage. Our study provides important insights into the origin and evolution of plant specialized metabolites, which are derived by duplication events of biosynthetic genes.

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.

Efficient Mimics for Elucidating Zaxinone Biology and Promoting Agricultural Applications

Zaxinone is an apocarotenoid regulatory metabolite required for normal rice growth and development.In addition,zaxinone has a large application potential in agriculture,due to its growth-promoting activity and capability to alleviate infestation by the root parasitic plant Striga through decreasing strigolactone(SL)production.However,zaxinone is poorly accessible to the scientific community because of its laborious organic synthesis that impedes its further investigation and utilization.In this study,we developed easy-to-synthesize and highly efficient mimics of zaxinone(MiZax).We performed a structure-activity relationship study using a series of apocarotenoids distinguished from zaxinone by different structural features.Using the obtained results,we designed several phenyl-based compounds synthesized with a high-yield through a simple method.Activity tests showed that MiZax3 and MiZax5 exert zaxinone activity in rescuing root growth of a zaxinone-deficient rice mutant,promoting growth,and reducing SL content in roots and root exudates of wild-type plants.Moreover,these compounds were at least as efficient as zaxinone in suppressing transcript level of SL biosynthesis genes and in alleviating Striga infestation under greenhouse conditions,and did not negatively impact mycorrhization.Taken together,MiZax are a promising tool for elucidating zaxinone biology and investigating rice development,and suitable candidates for combating Striga and increasing crop growth.

Natural Variation in CCD4 Promoter Underpins Species-Specific Evolution of Red Coloration in Citrus Peel

Carotenoids and apocarotenoids act as phytohormones and volatile precursors that influence plant development and confer aesthetic and nutritional value critical to consumer preference.Citrus fruits display considerable natural variation in carotenoid and apocarotenoid pigments.In this study,using an integrated genetic approach we revealed that a 5;c/s-regulatory change at CCD4b encoding CAROTENOID CLEAVAGE DIOXYGENASE 4b is a major genetic determinant of natural variation in C3 0 apocarotenoids responsible for red coloration of citrus peel.Functional analyses demonstrated that in addition the known role in synthesizing 3-citraurin,CCD4b is also responsible for the production of another important C3 0 apocarotenoid pigment,p-citraurinene.Furthermore,analyses of the CCD4b promoter and transcripts from various citrus germplasm accessions established a tight correlation between the presence of a putative 5'c/s-regulatory enhancer within an MITE transposon and the enhanced allelic expression of CCD4b in C3 0 apocarotenoid-rich red-peeled accessions.Phylogenetic analysis provided further evidence that functional diversification of CCD4b and naturally occurring variation of the CCD4b promoter resulted in the stepwise evolution of red peels in mandarins and their hybrids.Taken together,our findings provide new insights into the genetic and evolutionary basis of apocarotenoid diversity in plants,and would facilitate breeding efforts that aim to improve the nutritional and aesthetic value of citrus and perhaps other fruit crops.