Janet P. Slovin

The genome of woodland strawberry ( Fragaria vesca )

The woodland strawberry, Fragaria vesca (2n = 2x = 14), is a versatile experimental plant system. This diminutive herbaceous perennial has a small genome (240 Mb), is amenable to genetic transformation and shares substantial sequence identity with the cultivated strawberry (Fragaria × ananassa) and other economically important rosaceous plants. Here we report the draft F. vesca genome, which was sequenced to ×39 coverage using second-generation technology, assembled de novo and then anchored to the genetic linkage map into seven pseudochromosomes. This diploid strawberry sequence lacks the large genome duplications seen in other rosids. Gene prediction modeling identified 34,809 genes, with most being supported by transcriptome mapping. Genes critical to valuable horticultural traits including flavor, nutritional value and flowering time were identified. Macrosyntenic relationships between Fragaria and Prunus predict a hypothetical ancestral Rosaceae genome that had nine chromosomes. New phylogenetic analysis of 154 protein-coding genes suggests that assignment of Populus to Malvidae, rather than Fabidae, is warranted.

Auxin Biosynthesis and Metabolism

Auxins function at the intersection between environmental and developmental cues and the response pathways that they trigger (Fig. 1). Auxin levels vary dramatically throughout the body and life of the plant, forming gradients that are a central component of its action (4, 5, 14, 20, 34). Accordingly, plants have evolved intricate regulatory networks with considerable redundancy and adaptive plasticity to maintain auxin levels in response to changing environmental and developmental conditions. We refer to this phenomenon as auxin homeostasis; specifically the biosynthesis, inactivation, transport, and inter-conversion pathways that regulate and maintain auxin levels.

Indole-3-Acetic Acid Biosynthesis in the Mutant Maize orange pericarp, a Tryptophan Auxotroph.

The maize mutant orange pericarp is a tryptophan auxotroph, which results from mutation of two unlinked loci of tryptophan synthase B. This mutant was used to test the hypothesis that tryptophan is the precursor to the plant hormone indole-3-acetic acid (IAA). Total IAA in aseptically grown mutant seedlings was 50 times greater than in normal seedlings. In mutant seedlings grown on media containing stable isotopelabeled precursors, IAA was more enriched than was tryptophan. No incorporation of label into IAA from tryptophan could be detected. These results establish that IAA can be produced de novo without tryptophan as an intermediate.

Two genetically discrete pathways convert tryptophan to auxin: more redundancy in auxin biosynthesis

The answer to the simple question of how plants make auxin has proven to be inordinately complex. Recent in planta studies in Arabidopsis have uncovered additional complexity in auxin biosynthesis. Two distinct pathways from tryptophan to the intermediate indoleacetaldoxime were identified. Genic, as well as functional redundancy, appear to be characteristic for auxin biosynthesis and plants might have evolved many different solutions for making and regulating auxin.

A gene encoding a protein modified by the phytohormone indoleacetic acid.

We show that the expression of an indole-3-acetic acid (IAA)-modified protein from bean seed, IAP1, is correlated to the developmental period of rapid growth during seed development. Moreover, this protein undergoes rapid degradation during germination. The gene for IAP1, the most abundant protein covalently modified by IAA (iap1, GenBank accession no. AF293023) was isolated and cloned from bush bean (Phaseolus vulgaris) seeds. The 957-bp sequence encodes a 35-kDa polypeptide. IAA-modified proteins represent a distinct class of conjugated phytohormones and appear in bean to be the major form of auxin in seeds. IAA proteins also are found at other stages of development in bean plants. Our immunological and analytical data suggest that auxin modification of a small class of proteins may be a feature common to many plants.

Floral Transcriptomes in Woodland Strawberry Uncover Developing Receptacle and Anther Gene Networks

Genome-wide gene expression analyses in strawberry flowers identified key regulatory genes of the developing receptacle and anthers. Flowers are reproductive organs and precursors to fruits and seeds. While the basic tenets of the ABCE model of flower development are conserved in angiosperms, different flowering plants exhibit different and sometimes unique characteristics. A distinct feature of strawberry (Fragaria spp.) flowers is the development of several hundreds of individual apocarpous (unfused) carpels. These individual carpels are arranged in a spiral pattern on the subtending stem tip, the receptacle. Therefore, the receptacle is an integral part of the strawberry flower and is of significant agronomic importance, being the precursor to strawberry fruit. Taking advantage of next-generation sequencing and laser capture microdissection, we generated different tissue- and stage-transcriptomic profiling of woodland strawberry (Fragaria vesca) flower development. Using pairwise comparisons and weighted gene coexpression network analysis, we identified modules of coexpressed genes and hub genes of tissue-specific networks. Of particular importance is the discovery of a developing receptacle-specific module exhibiting similar molecular features to those of young floral meristems. The strawberry homologs of a number of meristem regulators, including LOST MERISTEM and WUSCHEL, are identified as hub genes operating in the developing receptacle network. Furthermore, almost 25% of the F-box genes in the genome are transiently induced in developing anthers at the meiosis stage, indicating active protein degradation. Together, this work provides important insights into the molecular networks underlying strawberry’s unique reproductive developmental processes. This extensive floral transcriptome data set is publicly available and can be readily queried at the project Web site, serving as an important genomic resource for the plant biology research community.

Indole-3-Acetic Acid Metabolism in Lemna gibba Undergoes Dynamic Changes in Response to Growth Temperature

Auxin is the mobile signal controlling the rate of growth and specific aspects of the development of plants. It has been known for over a century that auxins act as the messenger linking plant development to specific environmental changes. An often overlooked aspect of how this is accomplished is the effect of the environment on metabolism of the major plant auxin, indole-3-acetic acid (IAA). We have studied the metabolism of IAA in relation to one environmental variable, growth temperature. The model system used was an inbred line of the aquatic monocot Lemna gibba G-3, 3F7-11 grown at temperatures ranging from 5°C to 35°C. IAA levels, the rate of IAA turnover, and the patterns of label incorporation from IAA precursors were measured using stable isotope-mass spectrometric techniques and were evaluated relative to growth at the experimental temperatures. IAA levels exhibited unusually high variability in plants grown at 15°C and 20°C. Turnover rates were quite rapid throughout the range of experimental temperatures except at 25°C, where IAA turnover was notably slower. These results suggest that a transition occurred over these temperatures for some aspect of IAA metabolism. Analysis of [15N]anthranilate and [2H5]tryptophan (Trp) incorporation into IAA showed that Trp-dependent biosynthesis predominated at 15°C; however, Trp-independent biosynthesis of IAA was the major route to IAA at 30°C. The effects of growth temperature on auxin levels have been reported previously, but no prior studies correlated these effects with which pathway becomes the primary one for IAA production.

Profiling polyphenols of two diploid strawberry (Fragaria vesca) inbred lines using UHPLC-HRMSn

Phenolic compounds in the fruits of two diploid strawberries (Fragaria vesca f. semperflorens) inbred lines-Ruegen F7-4 (a red-fruited genotype) and YW5AF7 (a yellow-fruited genotype) were characterised using ultra-high-performance liquid chromatography coupled with tandem high-resolution mass spectrometry (UHPLC-HRMS(n)). The changes of anthocyanin composition during fruit development and between Ruegen F7-4 and YW5AF7 were studied. About 67 phenolic compounds, including taxifolin 3-O-arabinoside, glycosides of quercetin, kaempferol, cyanidin, pelargonidin, peonidin, ellagic acid derivatives, and other flavonols were identified in these two inbred lines. Compared to the regular octoploid strawberry, unique phenolic compounds were found in F. vesca fruits, such as taxifolin 3-O-arabinoside (both) and peonidin 3-O-malonylglucoside (Ruegen F7-4). The results provide the basis for comparative analysis of polyphenolic compounds in yellow and red diploid strawberries, as well as with the cultivated octoploid strawberries.

Indole-3-acetic acid biosynthesis in Lemna gibba studied using stable isotope labeled anthranilate and tryptophan

IAA biosynthesis in many plants, including Lemna gibba, has been shown to involve at least two different pathways, one from tryptophan and a tryptophan-independent route. To study the kinetics of IAA biosynthesis in Lemna, we simultaneously measured the incorporation of label from [15N]-anthranilate and [2H5]-tryptophan into IAA by Lemna plants in short term feeding studies. The data show that label from anthranilate rapidly goes into IAA and tryptophan. Labeling of the IAA pool by [15N]-anthranilate slightly precedes labeling of the tryptophan pool, confirming that more than one route to IAA exists in these plants. Longer term feeding studies (5–25 h) suggest that exogenous tryptophan is used preferentially to label IAA as compared to tryptophan made by the plant. This is indicated by the fact that the IAA pool was more enriched than the tryptophan pool in [2H5]-label, but less enriched than the tryptophan pool in [15N] (which comes about by de novo synthesis of tryptophan from [15N]-anthranilate by the plant).

Selection and Characterization of [alpha]-Methyltryptophan-Resistant Lines of Lemna gibba Showing a Rapid Rate of Indole-3-Acetic Acid Turnover.

Turnover rate is an important aspect of the regulation of plant processes by plant growth substances. To study turnover of indole-3-acetic acid (IAA), two [alpha]-methyltryptophan-resistant lines (MTR1 and MTR2) of Lemna gibba were generated by nitrosomethyl urea treatment of an inbred line derived from L. gibba G-3. In this report we describe: (a) the development of a selection system using this near isogenic line of L. gibba; (b) techniques for chemical mutation of the lines and selection for [alpha]-methyltryptophan resistance; and (c) the partial characterization of the selected lines. MTR lines contained 3-fold higher levels of anthranilate synthase activity. The enzyme in the MTR lines required higher levels of tryptophan for feedback inhibition. MTR lines also contained 8-fold higher levels of tryptophan, 3-fold higher levels of free IAA, and similar levels of total IAA compared to the inbred line. Turnover rates in the inbred and selected lines were calculated, using the first-order rate equation, based on the decrease over time in isotopic enrichment of I3C6-IAA introduced into L. gibba during a 1-h pulse period. Isotope enrichment in IAA was determined by using gas chromatography-mass spectrometry. Both MTR lines had an approximately 10-fold higher rate of IAA turnover than the parent inbred line.