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.Christopher I. Cazzonelli, Marleen Vanstraelen, Sibu Simon, Kuide Yin, Ashley Carron-Arthur, Nazia Nisar, Gauri Tarle, Abby J. Cuttriss¤, Iain R. Searle, Eva Benkova, Ulrike Mathesius, Josette Masle, Jiří Friml, Barry J. Pogso

Carotenoids

Carotenoid pigments provide fruits and flowers with distinctive red, orange and yellow colours as well as a number of aromas, which make them commercially important in agriculture, food, health and the cosmetic industries. Carotenoids comprise a large family of C40 polyenes that are critical for the survival of plants and animals alike. β-carotene and its derivatives contain unmodified β-ionone groups, which serve as precursors for vitamin A and are therefore essential dietary components for mammals. Significant progress has been made towards producing staple food crops with elevated provitamin A carotenoids, an important first step in alleviating worldwide vitamin A deficiency. Recent insights into the regulatory processes that control carotenoid composition and content may further advance biofortification projects

Carotenoids

Carotenoid pigments provide fruits and flowers with distinctive red, orange and yellow colours as well as a number of aromas, which make them commercially important in agriculture, food, health and the cosmetic industries. Carotenoids comprise a large family of C40 polyenes that are critical for the survival of plants and animals alike. ß-carotene and its derivatives contain unmodified ß-ionone groups, which serve as precursors for vitamin A and are therefore essential dietary components for mammals. Significant progress has been made towards producing staple food crops with elevated provitamin A carotenoids, an important first step in alleviating worldwide vitamin A deficiency. Recent insights into the regulatory processes that control carotenoid composition and content may further advance biofortification projects

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

Metabolite Sorting of a Germplasm Collection Reveals the Hydroxylase3 Locus as a New Target for Maize Provitamin A Biofortification1[C][W][OA]

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 β-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 β-carotene relative to β-cryptoxanthin and 36% of the variation and 4-fold difference in absolute levels of β-carotene. A simple PCR assay to track and identify Hydroxylase3 alleles will be valuable for predicting nutritional content in genetically diverse cultivars found worldwide

Regulation of Carotenoid Composition and Shoot Branching in Arabidopsis by a Chromatin Modifying Histone Methyltransferase, SDG8[W]

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 <i>PIN6</i> expression during plant development.

<p>Representative transgenic reporter lines harbouring −1794 bp (pPIN6::GUS; A–N) of the <i>PIN6</i> promoter fused to GUS. GUS activity was observed early during germination in fresh seeds (A), dried seeds (B), the root apical meristem of seedlings (C; 1DAG), regions of asymmetric root growth (D; 2 DAG) and in the SAM (E; 3 DAG). The <i>PIN6</i> promoter enabled GUS expression during lateral root initiation (F), at the boundaries of developing root primordia (G to J) and in the lateral RAM (K), floral organ boundaries (L, M) and anthers (N). Scale bars for G–K are similar to F. O) to X) Auxin up regulates <i>PIN6</i> promoter activity. Scale bars for P-S and U–X are similar to O. Auxin treatment (2 hrs) of pPIN6::GUS transgenic lines with 10 µM 2,4-D (T–X) induces GUS expression in the SAM (T) and root (U and V), but not in the lateral root primordia (W and X). Controls (O–S) were treated with ethanol only. Epidermis (Ep), cortex (Co), endodermis (En) and Pericycle (Pe).</p

Characterisation of <i>pin6</i> mutant alleles.

<p>A) Schematic diagram of the PIN6 genomic DNA fragment showing exon/intron structure (ATG and TGA refer to translation start and stop codons, respectively). PIN6 T-DNA insertions and primer positions (See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0070069#pone.0070069.s005" target="_blank">Table S1</a> for sequences) are marked by grey arrows. The Salk_046393 (<i>pin6-2</i>), Salk_092831 (<i>pin6-4</i>), GABI-KAT_430B01 (<i>pin6-5</i>) and GK711C09 (<i>pin6-6</i>) T-DNA insertions interrupt intron 2, exon 6, exon 3 and intron 6, respectively. B) Primary root length at 9 DAG (n = 25). (C) The development stage distributions of lateral root primordia that have initiated (I–VII) or emerged (Em) were quantified at 9 DAG (n = 10). D) The density (number of primordia per cm of primary root) of lateral root primordia in early (I–IV) and later (V–Em) stages of development, as well as the total number of primordia (n = 10). Similar results were observed in separate experiments. Homogeneity groups according to Student’s <i>t</i> test (p<0.05) are denoted with same letters. Error bars represent standard error.</p

PIN6 perturbs inter-cellular auxin homeostasis in the stem, roots and cotyledons.

<p>A) Total transport of ³H-IAA through an inflorescence stem section. Total auxin transport was calculated by adding the radioactivity determined for each 3mm segment isolated from a 30mm stem section. Average±SE (n = 4–12 independent stem sections). B) Auxin transport profile showing movement of ³H-IAA through the inflorescence stem segments 3 hours after loading labelled auxin. PIN1-OE refers to a PIN1 overexpressing line. The auxin transport results presented are representative of at least three independent experiments. C) and D) Root growth assays indicate that PIN6-OE#1 is less sensitive to IAA and IAA-alanine when compared to WT (Student’s <i>t</i> test *p<0.05, n = 30 plants, Error bars represent standard deviation). E) to P) <i>PIN6</i> overexpression enhances auxin accumulation in specific tissues. Histochemical GUS staining of DR5::GUS activity in germinating seeds (E, H), 3 day old seedlings (F, I) and mature primary root tips (G, J). K) to P) GUS staining of auxin treated (200 nM NAA) root tissues from WT (Fig. 5K to 5M) and PIN6-OE#14 (Fig. 5N to 5P) harboring the DR5::GUS reporter. Where appropriate, asterisks indicate a statistically significant difference (Student’s <i>t</i> test p<0.05).</p