Identification of key sites determining the cofactor specificity and improvement of catalytic activity of a steroid 5ß-reductase from Capsella rubella.

Progesterone 5ß-reductases (P5ßRs) are involved in 5ß-cardenolide formation by stereo-specific reduction of the △4,5 double bond of steroid precursors. In this study a steroid 5ß-reductase was identified in Capsella rubella (CrSt5ßR1) and its function in steroid 5ß-reduction was validated experimentally. CrSt5ßR1 is capable of enantioselectively reducing the activated CC bond of broad substrates such as steroids and enones by using NADPH as a cofactor and therefore has the potential as a biocatalyst in organic synthesis. However, for industrial purposes the cheaper NADH is the preferred cofactor. By applying rational design based on literature and complementary mutagenesis strategies, we successfully identified two key amino acid residues determining the cofactor specificity of the enzyme. The R63 K mutation enables the enzyme to convert progesterone to 5ß-pregnane-3,20-dione with NADH as cofactor, whereas the wild-type CrSt5ßR1 is strictly NADPH-dependent. By further introducing the R64H mutation, the double mutant R63K_R64H of CrSt5ßR1 was shown to increase enzymatic activity by13.8-fold with NADH as a cofactor and to increase the NADH/NADPH conversion ratio by 10.9-fold over the R63 K single mutant. This finding was successfully applied to change the cofactor specificity and to improve activity of other members of the same enzyme family, AtP5ßR and DlP5ßR. CrSt5ßR1 mutants are expected to have the potential for biotechnological applications in combination with the well-established NADH regeneration systems.

Polymerase IV Plays a Crucial Role in Pollen Development in Capsella.

In Arabidopsis (Arabidopsis thaliana), DNA-dependent RNA polymerase IV (Pol IV) is required for the formation of transposable element (TE)-derived small RNA transcripts. These transcripts are processed by DICER-LIKE3 into 24-nucleotide small interfering RNAs (siRNAs) that guide RNA-directed DNA methylation. In the pollen grain, Pol IV is also required for the accumulation of 21/22-nucleotide epigenetically activated siRNAs, which likely silence TEs via post-transcriptional mechanisms. Despite this proposed role of Pol IV, its loss of function in Arabidopsis does not cause a discernible pollen defect. Here, we show that the knockout of NRPD1, encoding the largest subunit of Pol IV, in the Brassicaceae species Capsella (Capsella rubella), caused postmeiotic arrest of pollen development at the microspore stage. As in Arabidopsis, all TE-derived siRNAs were depleted in Capsella nrpd1 microspores. In the wild-type background, the same TEs produced 21/22-nucleotide and 24-nucleotide siRNAs; these processes required Pol IV activity. Arrest of Capsella nrpd1 microspores was accompanied by the deregulation of genes targeted by Pol IV-dependent siRNAs. TEs were much closer to genes in Capsella compared with Arabidopsis, perhaps explaining the essential role of Pol IV in pollen development in Capsella. Our discovery that Pol IV is functionally required in Capsella microspores emphasizes the relevance of investigating different plant models.

Resistance evolution and mechanisms to ALS-inhibiting herbicides in Capsella bursa-pastoris populations from China.

Capsella bursa-pastoris is a serious broadleaf weed in winter wheat fields in China. It has evolved high levels of resistance to acetolactate synthase (ALS) inhibiting herbicides and has caused substantial losses of wheat yield in recent years. We monitored the herbicide resistance of Capsella bursa-pastoris collected from 18 regions of Shandong Province in 2009, 2013 and 2017, respectively. Compared with the 2009 populations, the number of populations resistant to florasulam had increased in 2013 and 2017. Resistance to tribenuron-methyl increased in 2013, but decreased in 2017. The 2009 and 2013 populations developed resistance only to tribenuron-methyl, but some 2017 populations developed cross-resistance to imazethapyr and florasulam as well. Mutations in ALS (Pro-197-Thr/Ser/His/Arg/Leu/Gln) were identified in the 2009 and 2013 populations; however, two ALS mutations (Pro197 and/or Trp574) were identified in 2017 plants. Meanwhile, plants containing both point mutations (Pro197 + Trp574) were identified in the 2017 populations. This study demonstrated that target site gene mutations were the main reason for Capsella bursa-pastoris resistance to ALS-inhibiting herbicides. Although target-site mutation is the reason for resistance to ALS-inhibiting herbicides in Capsella bursa-pastoris, the resistance patterns and mutations identified have changed over time.

Isolation and expression of acetolactate synthase genes that have a rare mutation in shepherd's purse (Capsella bursa-pastoris (L.) Medik.).

Acetolactate synthase (ALS) inhibitor-resistant biotypes are the fastest growing class of herbicide-resistant weeds. Shepherd's purse (Capsella bursa-pastoris (L.) Medik.), a tetraploid species and one of the most troublesome weeds in wheat production, has evolved ALS inhibitor resistance. To confirm and characterize the resistance of shepherd's purse populations to ALS-inhibiting herbicides, whole-plant bioassays were conducted. To investigate the molecular basis of resistance in shepherd's purse, the ALS gene was sequenced and compared between susceptible (S) and resistant (R) biotypes. Two partial intronless ALS genes (ALS-1 and ALS-2) were identified, and two heterozygous mutations (CCT to TCT in ALS-1 and CCT to CAT in ALS-2) at position 197 (Pro197Ser and Pro197His) providing resistance were simultaneously found in a single plant in a resistant population. Our results confirmed that the resistant shepherd's purse population showed high-level resistance to tribenuron-methyl (RI = 59.8), pyroxsulam (RI = 38.7) and flucarbazone-Na (RI = 88.0). Quantitative reverse transcription-polymerase chain reaction (RT-qPCR) results suggested that the difference in ALS gene expression was small between S and R populations, which may be insufficient to cause herbicide resistance, and according to the results of in vitro ALS activity, insensitivity of ALS may be the main mechanism of high resistance to tribenuron-methyl in resistant populations.

Regulation of FATTY ACID ELONGATION1 expression and production in Brassica oleracea and Capsella rubella.

MAIN CONCLUSION: The contribution of variations in coding regions or promoters to the changes in FAE1 expression levels have be quantified and compared in parallel by specifically designed swapping constructs. FATTY ACID ELONGATION1 (FAE1) is a key gene in control of erucic acid synthesis in plant seeds. The expression of FAE1 genes in Brassica oleracea and Capsella rubella, representatives of high and low erucic acid species, respectively, was characterized to provide insight into the regulation of very long-chain fatty-acid biosynthesis in seeds. Virtually, no methylation was detected either in B. oleracea or in C. rubella, suggesting that modification of promoter methylation might not be a predominant mechanism. Swapping constructs were specifically designed to quantify and compare the contribution of variations in coding regions or promoters to the changes in FAE1 expression levels in parallel. A significantly higher fold change in erucic acid content was observed when swapping coding regions rather than when swapping promoters, indicating that the coding region is a major determinant of the catalytic power of ß-ketoacyl-CoA synthase proteins. Common motifs have been proposed as essential for the preservation of basic gene expression patterns, such as seed-specific expression. However, the occurrence of variation in common cis-elements or the presence of species-specific cis-elements might be plausible mechanisms for changes in the expression levels in different organisms. In addition, conflicting observations in previous reports associated with FAE1 expression are discussed, and we suggest that caution should be taken when selecting a plant transformation vector and in interpreting the results obtained from vectors carrying the CaMV 35S promoter.

Repeated Inactivation of the First Committed Enzyme Underlies the Loss of Benzaldehyde Emission after the Selfing Transition in Capsella.

The enormous species richness of flowering plants is at least partly due to floral diversification driven by interactions between plants and their animal pollinators [1, 2]. Specific pollinator attraction relies on visual and olfactory floral cues [3-5]; floral scent can not only attract pollinators but also attract or repel herbivorous insects [6-8]. However, despite its central role for plant-animal interactions, the genetic control of floral scent production and its evolutionary modification remain incompletely understood [9-13]. Benzenoids are an important class of floral scent compounds that are generated from phenylalanine via several enzymatic pathways [14-17]. Here we address the genetic basis of the loss of floral scent associated with the transition from outbreeding to selfing in the genus Capsella. While the outbreeding C. grandiflora emits benzaldehyde as a major constituent of its floral scent, this has been lost in the selfing C. rubella. We identify the Capsella CNL1 gene encoding cinnamate:CoA ligase as responsible for this variation. Population genetic analysis indicates that CNL1 has been inactivated twice independently in C. rubella via different novel mutations to its coding sequence. Together with a recent study in Petunia [18], this identifies cinnamate:CoA ligase as an evolutionary hotspot for mutations causing the loss of benzenoid scent compounds in association with a shift in the reproductive strategy of Capsella from pollination by insects to self-fertilization.

Evolution of the self-incompatibility system in the Brassicaceae: identification of S-locus receptor kinase (SRK) in self-incompatible Capsella grandiflora.

Self-incompatibility (SI) has been well studied in the genera Brassica and Arabidopsis, which have become models for investigation into the SI system. To understand the evolution of the SI system in the Brassicaceae, comparative analyses of the S-locus in genera other than Brassica and Arabidopsis are necessary. We report the identification of six putative S-locus receptor kinase genes (SRK) in natural populations of Capsella grandiflora, an SI species from a genus which is closely related to Arabidopsis. These S-alleles display striking similarities to the Arabidopsis lyrata SRK alleles in sequence and structure. Our phylogenetic analysis supports the scenario of differing SI evolution along the two lineages (The Brassica lineage and Arabidopsis/Capsella lineage). Our results also argue that the ancestral S-locus lacked the SLG gene (S-locus glycoprotein) and that the diversification of S-alleles predates the separation of Arabidopsis and Capsella.