Genomic selection in plant breeding: Key factors shaping two decades of progress.

Genomic selection, the application of genomic prediction (GP) models to select candidate individuals, has significantly advanced in the past two decades, effectively accelerating genetic gains in plant breeding. This article provides a holistic overview of key factors that have influenced GP in plant breeding during this period. We delved into the pivotal roles of training population size and genetic diversity, and their relationship with the breeding population, in determining GP accuracy. Special emphasis was placed on optimizing training population size. We explored its benefits and the associated diminishing returns beyond an optimum size. This was done while considering the balance between resource allocation and maximizing prediction accuracy through current optimization algorithms. The density and distribution of single-nucleotide polymorphisms, level of linkage disequilibrium, genetic complexity, trait heritability, statistical machine-learning methods, and non-additive effects are the other vital factors. Using wheat, maize, and potato as examples, we summarize the effect of these factors on the accuracy of GP for various traits. The search for high accuracy in GP-theoretically reaching one when using the Pearson's correlation as a metric-is an active research area as yet far from optimal for various traits. We hypothesize that with ultra-high sizes of genotypic and phenotypic datasets, effective training population optimization methods and support from other omics approaches (transcriptomics, metabolomics and proteomics) coupled with deep-learning algorithms could overcome the boundaries of current limitations to achieve the highest possible prediction accuracy, making genomic selection an effective tool in plant breeding.

Novel sources of drought tolerance in sorghum landraces revealed via the analyses of genotype-by-environment interactions.

Globally, sorghum is the fifth most important crop, which is used for food, feed and fuel. However, its production and productivity are severely limited by various stresses, including drought. Hence, this study aimed to determine the responses of different drought-tolerance related traits in the Ethiopian sorghum germplasm through multi-environment field trials, thereby identifying novel sources of germplasm that can be used for breeding the crop for drought-tolerance. Three hundred twenty sorghum landraces and four improved varieties were grown at three sites within drought-prone areas (Melkassa, Mieso and Mehoni) in Ethiopia. The targeted traits were chlorophyll content at flowering (CHLF), chlorophyll content at maturity (CHLM), green leaf number at flowering (GLNF), stay-green (SG), flag leaf area (FLA), peduncle length (PDL), and panicle exertion (PAE). Multi-variate analyses of the collected data revealed the presence of high phenotypic variation in all traits. The combined and AMMI Analysis of variance showed that phenotypic variation due to the genotypes was higher for SG, CHLM, CHLF and GLNF and lower for FLA, PE and PDL in comparison with variation due to the environments or genotype by environment interactions. High broad sense heritability was observed for CHLF, CHLM, SG, GLNF, FLA, and PDL, whereas PAE showed moderate heritability. Due to the high heritability of chlorophyll content and the relatively small effect of environmental factors on it, it could serve as a criterion for selecting desirable genotypes for drought-tolerant breeding in sorghum. It has been found that chlorophyll content has a significant positive correlation with stay-green and grain yield, indicating that high chlorophyll content contributes to increasing grain yield by delaying the process of leaf senescence. The analyses of AMMI, GGE biplot, and genotype selection index revealed that several sorghum landraces outperformed the improved varieties with respect to CHLF, CHLM, and SG. Such landraces could serve as novel sources of germplasm for improving drought tolerance through breeding.

Genome-wide analyses using multi-locus models revealed marker-trait associations for major agronomic traits in Sorghum bicolor.

Globally, sorghum is the fifth most important cereal crop, and it is a major crop in Ethiopia, where it has a high genetic diversity. The country's sorghum gene pool contributes significantly to sorghum improvement worldwide. This study aimed to identify genomic regions and candidate genes associated with major agronomic traits in sorghum by using its genetic resources in Ethiopia for a genome-wide association study (GWAS). Phenotypic data of days to flowering (DTF), plant height (PH), panicle length (PALH), panicle width (PAWD), panicle weight (PAWT), and grain yield (GY) were collected from a GWAS panel comprising 324 sorghum accessions grown in three environments. SeqSNP, a targeted genotyping method, was used to genotype the panel using 5,000 gene-based single nucleotide polymorphism (SNP) markers. For marker-trait association (MTA) analyses, fixed and random model circulating probability unification (FarmCPU), and Bayesian-information and linkage-disequilibrium iteratively nested keyway (BLINK) models were used. In all traits, high phenotypic variation was observed, with broad-sense heritability ranging from 0.32 (for GY) to 0.90 (for PALH). A population structure, principal component analysis, and kinship analysis revealed that the accessions could be divided into two groups. In total, 54 MTAs were identified, 11 of which were detected by both BLINK and farmCPU. MTAs identified for each trait ranged from five (PAWT and GY) to fourteen (PH) representing both novel and previously identified quantitative trait loci (QTLs). Three SNPs were associated with more than one trait, including a SNP within the Sobic.004G189200 gene that was associated with PH and PAWT. Major effect SNP loci, Sbi2393610 (PVE = 23.3%), Sbi10438246 (PVE = 35.2%), Sbi17789352 (PVE = 11.9%) and Sbi30169733 (PVE = 18.9%) on chromosomes 1, 3, 5 and 9 that showed strong association signals for PAWD, DTF, GY and PALH, respectively, were major findings of this study. The SNP markers and candidate genes identified in this study provide insights into the genetic control of grain yield and related agronomic traits, and once validated, the markers could be used in genomics-led breeding.

A seed-like proteome in oil-rich tubers.

There are numerous examples of plant organs or developmental stages that are desiccation-tolerant and can withstand extended periods of severe water loss. One prime example are seeds and pollen of many spermatophytes. However, in some plants, also vegetative organs can be desiccation-tolerant. One example are the tubers of yellow nutsedge (Cyperus esculentus), which also store large amounts of lipids similar to seeds. Interestingly, the closest known relative, purple nutsedge (Cyperus rotundus), generates tubers that do not accumulate oil and are not desiccation-tolerant. We generated nanoLC-MS/MS-based proteomes of yellow nutsedge in five replicates of four stages of tuber development and compared them to the proteomes of roots and leaves, yielding 2257 distinct protein groups. Our data reveal a striking upregulation of hallmark proteins of seeds in the tubers. A deeper comparison to the tuber proteome of the close relative purple nutsedge (C. rotundus) and a previously published proteome of Arabidopsis seeds and seedlings indicates that indeed a seed-like proteome was found in yellow but not purple nutsedge. This was further supported by an analysis of the proteome of a lipid droplet-enriched fraction of yellow nutsedge, which also displayed seed-like characteristics. One reason for the differences between the two nutsedge species might be the expression of certain transcription factors homologous to ABSCISIC ACID INSENSITIVE3, WRINKLED1, and LEAFY COTYLEDON1 that drive gene expression in Arabidopsis seed embryos.

Transcriptional Regulation of Quinoa Seed Quality: Identification of Novel Candidate Genetic Markers for Increased Protein Content.

Quinoa (Chenopodium quinoa Willd.) is a crop that has great potential for increased cultivation in diverse climate regions. The seed protein quality obtained from this crop is high concerning the requirements to meet human nutritional needs, but the seed protein content is relatively low if compared to crops such as grain legumes. Increased seed protein content is desirable for increasing the economic viability of this crop in order for it to be used as a protein crop. In this study, we characterized three genotypes of quinoa with different levels of seed protein content. By performing RNA sequencing of developing seeds, we determined the genotype differences in gene expression and identified genetic polymorphisms that could be associated with increased protein content. Storage nutrient analyses of seeds of three quinoa genotypes (Titicaca, Pasankalla, and Regalona) from different ecoregions grown under controlled climate conditions showed that Pasankalla had the highest protein content (20%) and the lowest starch content (46%). Our seed transcriptome analyses revealed highly differentially expressed transcripts (DETs) in Pasankalla as compared to the other genotypes. These DETs encoded functions in sugar transport, starch and protein synthesis, genes regulating embryo size, and seed transcription factors. We selected 60 genes that encode functions in the central carbon metabolism and transcription factors as potential targets for the development of high-precision markers. Genetic polymorphisms, such as single nucleotide polymorphisms (SNPs) and base insertions and deletions (InDels), were found in 19 of the 60 selected genes, which can be further evaluated for the development of genetic markers for high seed protein content in quinoa. Increased cultivation of quinoa can contribute to a more diversified agriculture and support the plant protein diet shift. The identification of quinoa genotypes with contrasting seed quality can help establish a model system that can be used for the identification of precise breeding targets to improve the seed quality of quinoa. The data presented in this study based on nutrient and transcriptome analyses contribute to an enhanced understanding of the genetic regulation of seed quality traits in quinoa and suggest high-precision candidate markers for such traits.

Sorghum in dryland: morphological, physiological, and molecular responses of sorghum under drought stress.

MAIN CONCLUSION: Droughts negatively affect sorghum's productivity and nutritional quality. Across its diversity centers, however, there exist resilient genotypes that function differently under drought stress at various levels, including molecular and physiological. Sorghum is an economically important and a staple food crop for over half a billion people in developing countries, mostly in arid and semi-arid regions where drought stress is a major limiting factor. Although sorghum is generally considered tolerant, drought stress still significantly hampers its productivity and nutritional quality across its major cultivation areas. Hence, understanding both the effects of the stress and plant response is indispensable for improving drought tolerance of the crop. This review aimed at enhancing our understanding and provide more insights on drought tolerance in sorghum as a contribution to the development of climate resilient sorghum cultivars. We summarized findings on the effects of drought on the growth and development of sorghum including osmotic potential that impedes germination process and embryonic structures, photosynthetic rates, and imbalance in source-sink relations that in turn affect seed filling often manifested in the form of substantial reduction in grain yield and quality. Mechanisms of sorghum response to drought-stress involving morphological, physiological, and molecular alterations are presented. We highlighted the current understanding about the genetic basis of drought tolerance in sorghum, which is important for maximizing utilization of its germplasm for development of improved cultivars. Furthermore, we discussed interactions of drought with other abiotic stresses and biotic factors, which may increase the vulnerability of the crop or enhance its tolerance to drought stress. Based on the research reviewed in this article, it appears possible to develop locally adapted cultivars of sorghum that are drought tolerant and nutrient rich using modern plant breeding techniques.

Genotype by environment interaction, correlation, AMMI, GGE biplot and cluster analysis for grain yield and other agronomic traits in sorghum (Sorghum bicolor L. Moench).

Genotype by environment (G×E) interaction is a major factor limiting the success of germplasm selection and identification of superior genotypes for use in plant breeding programs. Similar to the case in other crops, G×E complicates the improvement of sorghum, and hence it should be determined and used in decision-making programs. The present study aimed at assessing the G×E interaction, and the correlation between traits for superior sorghum genotypes. Three hundred twenty sorghum landraces and four improved varieties were used in alpha lattice experimental design-based field trial across three environments (Melkassa, Mieso and Mehoni) in Ethiopia. Phenotypic data were collected for days to flowering (DTF), plant height (PH), panicle length (PALH), panicle width (PAWD), panicle weight (PAWT) and grain yield (GY). The results revealed that the variance due to genotype, environment and G×E interaction were highly significant (P < 0.001) for all traits. GY and PAWT were highly affected by environments and G×E whereas DTF, PALH, PAWD and PH were mainly affected by genotypic variation. Therefore, multi-environment testing is needed for taking care of G × E interaction to identify high yielding and stable sorghum landraces. GY and PAWT revealed highly significant positive correlations indicating the possibility of effective selection of the two traits simultaneously. Among the studied populations, South Wello, West Hararghe and Shewa zones had highly diverse genotypes that were distributed across all clusters. Hence, these areas can be considered as hotspots for identifying divergent sorghum landraces that could be used in breeding programs. Melkassa was the most representative environment whereas Mieso was the most discriminating. Five genotypes (G148, G123, G110, G203 and G73) were identified as superior across the test environments for grain yield with farmer-preferred trait, such as plant height. The identified stable and high yielding genotypes are valuable genetic resources that should be used in sorghum breeding programs.

Understanding the Sorghum-Colletotrichum sublineola Interactions for Enhanced Host Resistance.

Improving sorghum resistance is a sustainable method to reduce yield losses due to anthracnose, a devastating disease caused by Colletotrichum sublineola. Elucidating the molecular mechanisms of sorghum-C. sublineola interactions would help identify biomarkers for rapid and efficient identification of novel sources for host-plant resistance improvement, understanding the pathogen virulence, and facilitating resistance breeding. Despite concerted efforts to identify resistance sources, the knowledge about sorghum-anthracnose interactions remains scanty. Hence, in this review, we presented an overview of the current knowledge on the mechanisms of sorghum-C. sublineola molecular interactions, sources of resistance for sorghum breeding, quantitative trait loci (QTL), and major (R-) resistance gene sequences as well as defense-related genes associated with anthracnose resistance. We summarized current knowledge about C. sublineola populations and its virulence. Illustration of the sorghum-C. sublineola interaction model based on the current understanding is also provided. We highlighted the importance of genomic resources of both organisms for integrated omics research to unravel the key molecular components underpinning compatible and incompatible sorghum-anthracnose interactions. Furthermore, sorghum-breeding strategy employing rapid sorghum germplasm screening, systems biology, and molecular tools is presented.

Subcellular Localization of Acyl-CoA: Lysophosphatidylethanolamine Acyltransferases (LPEATs) and the Effects of Knocking-Out and Overexpression of Their Genes on Autophagy Markers Level and Life Span of A. thaliana.

Arabidopsis thaliana possesses two acyl-CoA:lysophosphatidylethanolamine acyltransferases, LPEAT1 and LPEAT2, which are encoded by At1g80950 and At2g45670 genes, respectively. Both single lpeat2 mutant and double lpeat1 lpeat2 mutant plants exhibit a variety of conspicuous phenotypes, including dwarfed growth. Confocal microscopic analysis of tobacco suspension-cultured cells transiently transformed with green fluorescent protein-tagged versions of LPEAT1 or LPEAT2 revealed that LPEAT1 is localized to the endoplasmic reticulum (ER), whereas LPEAT2 is localized to both Golgi and late endosomes. Considering that the primary product of the reaction catalyzed by LPEATs is phosphatidylethanolamine, which is known to be covalently conjugated with autophagy-related protein ATG8 during a key step of the formation of autophagosomes, we investigated the requirements for LPEATs to engage in autophagic activity in Arabidopsis. Knocking out of either or both LPEAT genes led to enhanced accumulation of the autophagic adaptor protein NBR1 and decreased levels of both ATG8a mRNA and total ATG8 protein. Moreover, we detected significantly fewer membrane objects in the vacuoles of lpeat1 lpeat2 double mutant mesophyll cells than in vacuoles of control plants. However, contrary to what has been reported on autophagy deficient plants, the lpeat mutants displayed a prolonged life span compared to wild type, including delayed senescence.

Comparison of Morphological and Genetic Characteristics of Avocados Grown in Tanzania.

Tanzania has been growing avocado for decades. A wide variability of the avocado germplasm has been found, and the crop is largely contributing to the earnings of the farmers, traders, and the government, but its genetic diversity is scantly investigated. With the purpose of comparing morphological and genetic characteristics of this germplasm and uncovering the correlation between them and the geographical location, 226 adult seedling avocado trees were sampled in southwestern Tanzania. Their morphological characters were recorded, and their genetic diversity was evaluated based on 10 microsatellite loci. Discriminant analysis of principal components showed that the germplasm studied consisted of four genetic clusters that had an overall average gene diversity of 0.59 and 15.9% molecular variation among them. Most of the phenotypes were common in at least two clusters. The genetic clusters were also portrayed by multivariate analysis and hierarchical clustering for the molecular data but not for the morphology data. Using the Mantel test, a weak significant correlation was found between the genetic, morphological, and geographical distances, which indicates that the genetic variation present in the material is weakly reflected by the observed phenotypic variation and that both measures of variation varied slightly with the geographical sampling locations.

Genetic Diversity and Population Structure of Sorghum [Sorghum Bicolor (L.) Moench] Accessions as Revealed by Single Nucleotide Polymorphism Markers.

Ethiopia is the center of origin for sorghum [Sorghum bicolor (L.) Moench], where the distinct agro-ecological zones significantly contributed to the genetic diversity of the crops. A large number of sorghum landrace accessions have been conserved ex situ. Molecular characterization of this diverse germplasm can contribute to its efficient conservation and utilization in the breeding programs. This study aimed to investigate the genetic diversity of Ethiopian sorghum using gene-based single nucleotide polymorphism (SNP) markers. In total, 359 individuals representing 24 landrace accessions were genotyped using 3,001 SNP markers. The SNP markers had moderately high polymorphism information content (PIC = 0.24) and gene diversity (H = 0.29), on average. This study revealed 48 SNP loci that were significantly deviated from Hardy-Weinberg equilibrium with excess heterozygosity and 13 loci presumed to be under selection (P < 0.01). The analysis of molecular variance (AMOVA) determined that 35.5% of the total variation occurred within and 64.5% among the accessions. Similarly, significant differentiations were observed among geographic regions and peduncle shape-based groups. In the latter case, accessions with bent peduncles had higher genetic variation than those with erect peduncles. More alleles that are private were found in the eastern region than in the other regions of the country, suggesting a good in situ conservation status in the east. Cluster, principal coordinates (PCoA), and STRUCTURE analyses revealed distinct accession clusters. Hence, crossbreeding genotypes from different clusters and evaluating their progenies for desirable traits is advantageous. The exceptionally high heterozygosity observed in accession SB4 and SB21 from the western geographic region is an intriguing finding of this study, which merits further investigation.

Genetic diversity of avocado from the southern highlands of Tanzania as revealed by microsatellite markers.

BACKGROUND: Avocado is an important cash crop in Tanzania, however its genetic diversity is not thoroughly investigated. This study was undertaken to explore the genetic diversity of avocado in the southern highlands using microsatellite markers. A total of 226 local avocado trees originating from seeds were sampled in eight districts of the Mbeya, Njombe and Songwe regions. Each district was considered as a population. The diversity at 10 microsatellite loci was investigated. RESULTS: A total of 167 alleles were detected across the 10 loci with an average of 16.7 ± 1.3 alleles per locus. The average expected and observed heterozygosity were 0.84 ± 0.02 and 0.65 ± 0.04, respectively. All but two loci showed a significant deviation from the Hardy-Weinberg principle. Analysis of molecular variance showed that about 6% of the variation was partitioned among the eight geographic populations. Population FST pairwise comparisons revealed lack of genetic differentiation for the seven of 28 population pairs tested. The principal components analysis (PCA) and hierarchical cluster analysis showed a mixing of avocado trees from different districts. The model-based STRUCTURE subdivided the trees samples into four major genetic clusters. CONCLUSION: High diversity detected in the analysed avocado germplasm implies that this germplasm is a potentially valuable source of variable alleles that might be harnessed for genetic improvement of this crop in Tanzania. The mixing of avocado trees from different districts observed in the PCA and dendrogram points to strong gene flow among the avocado populations, which led to population admixture revealed in the STRUCTURE analysis. However, there is still significant differentiation among the tree populations from different districts that can be utilized in the avocado breeding program.

Transitions in wheat endosperm metabolism upon transcriptional induction of oil accumulation by oat endosperm WRINKLED1.

BACKGROUND: Cereal grains, including wheat (Triticum aestivum L.), are major sources of food and feed, with wheat being dominant in temperate zones. These end uses exploit the storage reserves in the starchy endosperm of the grain, with starch being the major storage component in most cereal species. However, oats (Avena sativa L.) differs in that the starchy endosperm stores significant amounts of oil. Understanding the control of carbon allocation between groups of storage compounds, such as starch and oil, is therefore important for understanding the composition and hence end use quality of cereals. WRINKLED1 is a transcription factor known to induce triacylglycerol (TAG; oil) accumulation in several plant storage tissues. RESULTS: An oat endosperm homolog of WRI1 (AsWRI1) expressed from the endosperm-specific HMW1Dx5 promoter resulted in drastic changes in carbon allocation in wheat grains, with reduced seed weight and a wrinkled seed phenotype. The starch content of mature grain endosperms of AsWRI1-wheat was reduced compared to controls (from 62 to 22% by dry weight (dw)), TAG was increased by up to nine-fold (from 0.7 to 6.4% oil by dw) and sucrose from 1.5 to 10% by dw. Expression of AsWRI1 in wheat grains also resulted in multiple layers of elongated peripheral aleurone cells. RNA-sequencing, lipid analyses, and pulse-chase experiments using 14C-sucrose indicated that futile cycling of fatty acids could be a limitation for oil accumulation. CONCLUSIONS: Our data show that expression of oat endosperm WRI1 in the wheat endosperm results in changes in metabolism which could underpin the application of biotechnology to manipulate grain composition. In particular, the striking effect on starch synthesis in the wheat endosperm indicates that an important indirect role of WRI1 is to divert carbon allocation away from starch biosynthesis in plant storage tissues that accumulate oil.

Crambe hispanica Subsp. abyssinica Diacylglycerol Acyltransferase Specificities Towards Diacylglycerols and Acyl-CoA Reveal Combinatorial Effects That Greatly Affect Enzymatic Activity and Specificity.

Crambe is an oil crop suitable for industrial purposes due to the high content of erucic acid (22:1) in the seed oil. The final acylation of diacylglycerols (DAG) with acyl-CoA in the production of triacylglycerols (oil) is catalyzed by acyl-CoA:diacylglycerol acyltransferase (DGAT) enzymes. We identified eight forms of DGATs in crambe and characterized them in microsomal preparations of yeast expressing the enzymes using various acyl-CoAs and both di-6:0-DAG and long-chain DAG species as acyl acceptors. All DGATs accepted 22:1-CoA when using di-6:0-DAG as acyl acceptor. When di-22:1-DAG was the acyl acceptor, the DGAT1 type of enzyme utilized 22:1-CoA at a much-reduced rate compared to assays with sn-1-22:1-sn-2-18:1(oleoyl)-DAG, the most frequently available DAG precursor in crambe seeds. None of the DGAT2 enzymes was able to acylate di-22:1-DAG. Our results indicate that formation of trierucin by crambe DGATs is a limiting step for further increasing the levels of 22:1 in the previously developed transgenic crambe lines due to their poor abilities to acylate di-22:1-DAG. We also show that the acyl-CoA specificities and the enzymatic activities are highly influenced by the fatty acid composition of the DAG acyl acceptor. This finding implies that the use of artificial acyl acceptors (e.g. di-6:0-DAG) may not always reflect the actual acyl-CoA specificities of DGATs in planta. The relevance of the here reported pronounced specificities for specific DAG species exerted by DGAT enzymes is discussed in the context of the findings of DAG pools of distinct catalytic origin in triacylglycerol biosynthesis in the seed oil.

Charting oat (Avena sativa) embryo and endosperm transcription factor expression reveals differential expression of potential importance for seed development.

Uniquely, oat, among cereals, accumulates an appreciable amount of oil in the endosperm together with starch. Oat is also recognized for its soluble fibers in the form of ß-glucans. Despite high and increasing interest in oat yield and quality, the genetic and molecular understanding of oat grain development is still very limited. Transcription factors (TFs) are important regulatory components for plant development, product quality and yield. This study aimed to develop a workflow to determine seed tissue specificity of transcripts encoding transcription factors to reveal differential expression of potential importance for storage compound deposition and quality characters in oat. We created a workflow through the de novo assembly of sequenced seed endosperm and embryo, and publicly available oat seed RNAseq dataset, later followed by TF identification. RNAseq data were assembled into 33,878 transcripts with approximately 90% completeness. A total of 3875 putative TF encoding transcripts were identified from the oat hybrid assemblies. Members of the B3, bHLH, bZIP, C3H, ERF, NAC, MYB and WRKY families were the most abundant TF transcripts. A total of 514 transcripts which were differentially expressed between embryo and endosperm were identified with a threshold of 16-fold expression difference. Among those, 36 TF transcript homologs, belonging to 7 TF families, could be identified through similarity search in wheat embryo and endosperm EST libraries of NCBI Unigene database, and almost all the closest homologs were specifically expressed in seed when explored in WheatExp database. We verified our findings by cloning, sequencing and finally confirming differential expression of two TF encoding transcripts in oat seed embryo and endosperm. The developed workflow for identifying tissue-specific transcription factors allows further functional characterization of specific genes to increase our understanding of grain filling and quality.

WRINKLED1 Is Subject to Evolutionary Conserved Negative Autoregulation.

High accumulation of storage compounds such as oil and starch are economically important traits of most agricultural crops. The genetic network determining storage compounds composition in crops has been the target of many biotechnological endeavors. Especially WRINKLED1 (WRI1), a well-known key transcription factor involved in the allocation of carbon into oil, has attracted much interest. Here we investigate the presence of an autoregulatory system involving WRI1 through transient expression in Nicotiana benthamiana leaves. Different lengths of the Arabidopsis WRI1 promotor region were coupled to a GUS reporter gene and the activity was measured when combined with constitutive expression of different WRI1 homologs from Arabidopsis thaliana, oat (Avena sativa L.), yellow nutsedge (Cyperus esculentus L.), and potato (Solanum tuberosum L.). We could show that increasing levels of each WRI1 homolog reduced the transcriptional activity of the Arabidopsis WRI1 upstream region. Through structural analysis and domain swapping between oat and Arabidopsis WRI1, we were able to determine that the negative autoregulation was clearly dependent on the DNA-binding AP2-domains. A DNA/protein interaction assay showed that AtWRI1 is unable to bind to its corresponding upstream region indicating non-direct interaction in vivo. Taken together, our results demonstrate a negative feedback loop of WRI1 expression and that it is an indirect interaction most likely caused by downstream targets of WRI1. We also show that it is possible to release WRI1 expression from this autoregulation by creating semi-synthetic WRI1 homologs increasing the potential use of WRI1 in biotechnological applications.

Acyl-CoA:Lysophosphatidylethanolamine Acyltransferase Activity Regulates Growth of Arabidopsis.

Arabidopsis (Arabidopsis thaliana) contains two enzymes (encoded by the At1g80950 and At2g45670 genes) preferentially acylating lysophosphatidylethanolamine (LPE) with acyl-coenzyme A (CoA), designated LYSOPHOSPHATIDYLETHANOLAMINE ACYLTRANSFERASE1 (LPEAT1) and LPEAT2. The transfer DNA insertion mutant lpeat2 and the double mutant lpeat1 lpeat2 showed impaired growth, smaller leaves, shorter roots, less seed setting, and reduced lipid content per fresh weight in roots and seeds and large increases in LPE and lysophosphatidylcholine (LPC) contents in leaves. Microsomal preparations from leaves of these mutants showed around 70% decrease in acylation activity of LPE with 16:0-CoA compared with wild-type membranes, whereas the acylation with 18:1-CoA was much less affected, demonstrating that other lysophospholipid acyltransferases than the two LPEATs could acylate LPE The above-mentioned effects were less pronounced in the single lpeat1 mutant. Overexpression of either LPEAT1 or LPEAT2 under the control of the 35S promotor led to morphological changes opposite to what was seen in the transfer DNA mutants. Acyl specificity studies showed that LPEAT1 utilized 16:0-CoA at the highest rate of 11 tested acyl-CoAs, whereas LPEAT2 utilized 20:0-CoA as the best acyl donor. Both LPEATs could acylate either sn position of ether analogs of LPC The data show that the activities of LPEAT1 and LPEAT2 are, in a complementary way, involved in growth regulation in Arabidopsis. It is shown that LPEAT activity (especially LPEAT2) is essential for maintaining adequate levels of phosphatidylethanolamine, LPE, and LPC in the cells.

Biochemical characteristics of AtFAR2, a fatty acid reductase from Arabidopsis thaliana that reduces fatty acyl-CoA and -ACP substrates into fatty alcohols.

Fatty alcohols and derivatives are important for proper deposition of a functional pollen wall. Mutations in specific genes encoding fatty acid reductases (FAR) responsible for fatty alcohol production cause abnormal development of pollen. A disrupted AtFAR2 (MS2) gene in Arabidopsis thaliana results in pollen developing an abnormal exine layer and a reduced fertility phenotype. AtFAR2 has been shown to be targeted to chloroplasts and in a purified form to be specific for acyl-ACP substrates. Here, we present data on the in vitro and in planta characterizations of AtFAR2 from A. thaliana and show that this enzyme has the ability to use both, C16:0-ACP and C16:0-CoA, as substrates to produce C16:0-alcohol. Our results further show that AtFAR2 is highly similar in properties and substrate specificity to AtFAR6 for which in vitro data has been published, and which is also a chloroplast localized enzyme. This suggests that although AtFAR2 is the major enzyme responsible for exine layer functionality, AtFAR6 might provide functional redundancy to AtFAR2.

Potato tuber expression of Arabidopsis WRINKLED1 increase triacylglycerol and membrane lipids while affecting central carbohydrate metabolism.

Tuber and root crops virtually exclusively accumulate storage products in the form of carbohydrates. An exception is yellow nutsedge (Cyperus esculentus) in which tubers have the capacity to store starch and triacylglycerols (TAG) in roughly equal amounts. This suggests that a tuber crop can efficiently handle accumulation of energy dense oil. From a nutritional as well as economic aspect, it would be of interest to utilize the high yield capacity of tuber or root crops for oil accumulation similar to yellow nutsedge. The transcription factor WRINKLED1 from Arabidopsis thaliana, which in seed embryos induce fatty acid synthesis, has been shown to be a major factor for oil accumulation. WRINKLED1 was expressed in potato (Solanum tuberosum) tubers to explore whether this factor could impact tuber metabolism. This study shows that a WRINKLED1 transcription factor could induce triacylglycerol accumulation in tubers of transformed potato plants grown in field (up to 12 nmol TAG/mg dry weight, 1% of dry weight) together with a large increase in polar membrane lipids. The changes in metabolism further affected starch accumulation and composition concomitant with massive increases in sugar content.

Transcriptional transitions in Nicotiana benthamiana leaves upon induction of oil synthesis by WRINKLED1 homologs from diverse species and tissues.

BACKGROUND: Carbon accumulation and remobilization are essential mechanisms in plants to ensure energy transfer between plant tissues with different functions or metabolic needs and to support new generations. Knowledge about the regulation of carbon allocation into oil (triacylglycerol) in plant storage tissue can be of great economic and environmental importance for developing new high-yielding oil crops. Here, the effect on global gene expression as well as on physiological changes in leaves transiently expressing five homologs of the transcription factor WRINKLED1 (WRI1) originating from diverse species and tissues; Arabidopsis thaliana and potato (Solanum tuberosum) seed embryo, poplar (Populus trichocarpa) stem cambium, oat (Avena sativa) grain endosperm, and nutsedge (Cyperus esculentus) tuber parenchyma, were studied by agroinfiltration in Nicotiana benthamiana. RESULTS: All WRI1 homologs induced oil accumulation when expressed in leaf tissue. Transcriptome sequencing revealed that all homologs induced the same general patterns with a drastic shift in gene expression profiles of leaves from that of a typical source tissue to a source-limited sink-like tissue: Transcripts encoding enzymes for plastid uptake and metabolism of phosphoenolpyruvate, fatty acid and oil biosynthesis were up-regulated, as were also transcripts encoding starch degradation. Transcripts encoding enzymes in photosynthesis and starch synthesis were instead down-regulated. Moreover, transcripts representing fatty acid degradation were up-regulated indicating that fatty acids might be degraded to feed the increased need to channel carbons into fatty acid synthesis creating a futile cycle. RT-qPCR analysis of leaves expressing Arabidopsis WRI1 showed the temporal trends of transcripts selected as 'markers' for key metabolic pathways one to five days after agroinfiltration. Chlorophyll fluorescence measurements of leaves expressing Arabidopsis WRI1 showed a significant decrease in photosynthesis, even though effect on starch content could not be observed. CONCLUSIONS: This data gives for the first time a general view on the transcriptional transitions in leaf tissue upon induction of oil synthesis by WRI1. This yields important information about what effects WRI1 may exert on global gene expression during seed and embryo development. The results suggest why high oil content in leaf tissue cannot be achieved by solely transcriptional activation by WRI1, which can be essential knowledge in the development of new high-yielding oil crops.