Metabolite sorting of a germplasm collection reveals the hydroxylase3 locus as a new target for maize provitamin A biofortification.

Vitamin A deficiency, a global health burden, can be alleviated through provitamin A carotenoid biofortification of major crop staples such as maize (Zea mays) and other grasses in the Poaceae. If regulation of carotenoid biosynthesis was better understood, enhancement could be controlled by limiting beta-carotene hydroxylation to compounds with lower or no nonprovitamin A activity. Natural maize genetic diversity enabled identification of hydroxylation genes associated with reduced endosperm provitamin A content. A novel approach was used to capture the genetic and biochemical diversity of a large germplasm collection, representing 80% of maize genetic diversity, without having to sample the entire collection. Metabolite data sorting was applied to select a 10-line genetically diverse subset representing biochemical extremes for maize kernel carotenoids. Transcript profiling led to discovery of the Hydroxylase3 locus that coincidently mapped to a carotene quantitative trait locus, thereby prompting investigation of allelic variation in a broader collection. Three natural alleles in 51 maize lines explained 78% of variation and approximately 11-fold difference in beta-carotene relative to beta-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of beta-carotene. A simple PCR assay to track and identify Hydroxylase3 alleles will be valuable for predicting nutritional content in genetically diverse cultivars found worldwide.

The promoter of the Arabidopsis PIN6 auxin transporter enabled strong expression in the vasculature of roots, leaves, floral stems and reproductive organs.

Cellular auxin homeostasis controls many aspects of plant growth, organogenesis and development. The existence of intracellular auxin transport mediated by endoplasmic reticulum (ER)-localized PIN5, PIN6 and PIN8 proteins is a relatively recent discovery shaping a new era in understanding auxin-mediated growth processes. Here we summarize the importance of PIN6 in mediating intracellular auxin transport during root formation, leaf vein patterning and nectary production. While, it was previously shown that PIN6 was strongly expressed in rosette leaf cell types important in vein formation, here we demonstrate by use a PIN6 promoter-reporter fusion, that PIN6 is also preferentially expressed in the vasculature of the primary root, cotyledons, cauline leaves, floral stem, sepals and the main transmitting tract of the reproductive silique. The strong, vein- specific reporter gene expression patterns enabled by the PIN6 promoter emphasizes that transcriptional control is likely to be a major regulator of PIN6 protein levels, during vasculature formation, and supports the need for ER-localized PIN proteins in selecting specialized cells for vascular function in land plants.

Role of the Arabidopsis PIN6 auxin transporter in auxin homeostasis and auxin-mediated development.

Plant-specific PIN-formed (PIN) efflux transporters for the plant hormone auxin are required for tissue-specific directional auxin transport and cellular auxin homeostasis. The Arabidopsis PIN protein family has been shown to play important roles in developmental processes such as embryogenesis, organogenesis, vascular tissue differentiation, root meristem patterning and tropic growth. Here we analyzed roles of the less characterised Arabidopsis PIN6 auxin transporter. PIN6 is auxin-inducible and is expressed during multiple auxin-regulated developmental processes. Loss of pin6 function interfered with primary root growth and lateral root development. Misexpression of PIN6 affected auxin transport and interfered with auxin homeostasis in other growth processes such as shoot apical dominance, lateral root primordia development, adventitious root formation, root hair outgrowth and root waving. These changes in auxin-regulated growth correlated with a reduction in total auxin transport as well as with an altered activity of DR5-GUS auxin response reporter. Overall, the data indicate that PIN6 regulates auxin homeostasis during plant development.

Identifying chloroplast biogenesis and signalling mutants in Arabidopsis thaliana.

The chloroplast is the largest and arguably the most complex of the three energy organelles in the plant cell. The biogenesis of the chloroplast requires a combination of thousands of proteins encoded by the chloroplastic and nuclear genomes. Chloroplast function is also subject to modifications to enable responses to changes in environmental and developmental stimuli. As a consequence, interorganelle signalling and coordination between the chloroplast and nucleus is critical for the biogenesis and function of the chloroplast. Coordination and signalling during biogenesis is referred to as biogenic control and during the function as operational control (1). In this article, we report on two different mutant screens as examples of strategies for identifying mutations that affect biogenic and operational control signalling pathways and processes. We also describe strategies for the analysis and genotyping of the mutants.

Regulation of carotenoid composition and shoot branching in Arabidopsis by a chromatin modifying histone methyltransferase, SDG8.

Carotenoid pigments are critical for plant survival, and carotenoid composition is tuned to the developmental stage, tissue, and to environmental stimuli. We report the cloning of the CAROTENOID CHLOROPLAST REGULATORY1 (CCR1) gene. The ccr1 mutant has increased shoot branching and altered carotenoid composition, namely, reduced lutein in leaves and accumulation of cis-carotenes in dark-grown seedlings. The CCR1 gene was previously isolated as EARLY FLOWERING IN SHORT DAYS and encodes a histone methyltransferase (SET DOMAIN GROUP 8) that methylates histone H3 on Lys 4 and/or 36 (H3K4 and H3K36). ccr1 plants show reduced trimethyl-H3K4 and increased dimethyl-H3K4 surrounding the CAROTENOID ISOMERASE (CRTISO) translation start site, which correlates with low levels of CRTISO mRNA. Microarrays of ccr1 revealed the downregulation of 85 genes, including CRTISO and genes associated with signaling and development, and upregulation of just 28 genes. The reduction in CRTISO transcript abundance explains the altered carotenoid profile. The changes in shoot branching are additive with more axillary branching mutants, but the altered carotenoid profile may partially affect shoot branching, potentially by perturbed biosynthesis of the carotenoid substrates of strigolactones. These results are consistent with SDG8 regulating shoot meristem activity and carotenoid biosynthesis by modifying the chromatin surrounding key genes, including CRTISO. Thus, the level of lutein, the most abundant carotenoid in higher plants that is critical for photosynthesis and photoprotection, appears to be regulated by a chromatin modifying enzyme in Arabidopsis thaliana.

Regulation of lutein biosynthesis and prolamellar body formation in Arabidopsis.

Carotenoids are critical for photosynthetic function in chloroplasts, and are essential for the formation of the prolamellar body in the etioplasts of dark-grown (etiolated) seedlings. They are also precursors for plant hormones in both types of plastids. Lutein is one of the most abundant carotenoids found in both plastids. In this study we examine the regulation of lutein biosynthesis and investigate the effect of perturbing carotenoid biosynthesis on the formation of the lattice-like membranous structure of etioplasts, the prolamellar body (PLB). Analysis of mRNA abundance in wildtype and lutein-deficient mutants, lut2 and ccr2, in response to light transitions and herbicide treatments demonstrated that the mRNA abundance of the carotenoid isomerase (CRTISO) and epsilon-cyclase (ϵLCY) can be rate limiting steps in lutein biosynthesis. We show that accumulation of tetra-cis-lycopene and all-trans-lycopene correlates with the abundance of mRNA of several carotenoid biosynthetic genes. Herbicide treatments that inhibit carotenoid biosynthetic enzymes in wildtype and ccr2 etiolated seedlings were used to demonstrate that the loss of the PLB in ccr2 mutants is a result of perturbations in carotenoid accumulation, not indirect secondary effects, as PLB formation could be restored in ccr2 mutants treated with norflurazon.

Identification of the carotenoid isomerase provides insight into carotenoid biosynthesis, prolamellar body formation, and photomorphogenesis.

Carotenoids are essential photoprotective and antioxidant pigments synthesized by all photosynthetic organisms. Most carotenoid biosynthetic enzymes were thought to have evolved independently in bacteria and plants. For example, in bacteria, a single enzyme (CrtI) catalyzes the four desaturations leading from the colorless compound phytoene to the red compound lycopene, whereas plants require two desaturases (phytoene and zeta-carotene desaturases) that are unrelated to the bacterial enzyme. We have demonstrated that carotenoid desaturation in plants requires a third distinct enzyme activity, the carotenoid isomerase (CRTISO), which, unlike phytoene and zeta-carotene desaturases, apparently arose from a progenitor bacterial desaturase. The Arabidopsis CRTISO locus was identified by the partial inhibition of lutein synthesis in light-grown tissue and the accumulation of poly-cis-carotene precursors in dark-grown tissue of crtISO mutants. After positional cloning, enzymatic analysis of CRTISO expressed in Escherichia coli confirmed that the enzyme catalyzes the isomerization of poly-cis-carotenoids to all-trans-carotenoids. Etioplasts of dark-grown crtISO mutants accumulate acyclic poly-cis-carotenoids in place of cyclic all-trans-xanthophylls and also lack prolamellar bodies (PLBs), the lattice of tubular membranes that defines an etioplast. This demonstrates a requirement for carotenoid biosynthesis to form the PLB. The absence of PLBs in crtISO mutants demonstrates a function for this unique structure and carotenoids in facilitating chloroplast development during the first critical days of seedling germination and photomorphogenesis.