Redox regulation of a guard cell SNF1-related protein kinase in Brassica napus, an oilseed crop.

Kinase-mediated phosphorylation is a pivotal regulatory process in stomatal responses to stresses. Through a redox proteomics study, a sucrose non-fermenting 1-related protein kinase (SnRK2.4) was identified to be redox-regulated in Brassica napus guard cells upon abscisic acid treatment. There are six genes encoding SnRK2.4 paralogs in B. napus Here, we show that recombinant BnSnRK2.4-1C exhibited autophosphorylation activity and preferentially phosphorylated the N-terminal region of B. napus slow anion channel (BnSLAC1-NT) over generic substrates. The in vitro activity of BnSnRK2.4-1C requires the presence of manganese (Mn2+). Phosphorylation sites of autophosphorylated BnSnRK2.4-1C were mapped, including serine and threonine residues in the activation loop. In vitro BnSnRK2.4-1C autophosphorylation activity was inhibited by oxidants such as H2O2 and recovered by active thioredoxin isoforms, indicating redox regulation of BnSnRK2.4-1C. Thiol-specific isotope tagging followed by mass spectrometry analysis revealed specific cysteine residues responsive to oxidant treatments. The in vivo activity of BnSnRK2.4-1C is inhibited by 15 min of H2O2 treatment. Taken together, these data indicate that BnSnRK2.4-1C, an SnRK preferentially expressed in guard cells, is redox-regulated with potential roles in guard cell signal transduction.

A faster Rubisco with potential to increase photosynthesis in crops.

In photosynthetic organisms, D-ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) is the major enzyme assimilating atmospheric CO2 into the biosphere. Owing to the wasteful oxygenase activity and slow turnover of Rubisco, the enzyme is among the most important targets for improving the photosynthetic efficiency of vascular plants. It has been anticipated that introducing the CO2-concentrating mechanism (CCM) from cyanobacteria into plants could enhance crop yield. However, the complex nature of Rubisco's assembly has made manipulation of the enzyme extremely challenging, and attempts to replace it in plants with the enzymes from cyanobacteria and red algae have not been successful. Here we report two transplastomic tobacco lines with functional Rubisco from the cyanobacterium Synechococcus elongatus PCC7942 (Se7942). We knocked out the native tobacco gene encoding the large subunit of Rubisco by inserting the large and small subunit genes of the Se7942 enzyme, in combination with either the corresponding Se7942 assembly chaperone, RbcX, or an internal carboxysomal protein, CcmM35, which incorporates three small subunit-like domains. Se7942 Rubisco and CcmM35 formed macromolecular complexes within the chloroplast stroma, mirroring an early step in the biogenesis of cyanobacterial ß-carboxysomes. Both transformed lines were photosynthetically competent, supporting autotrophic growth, and their respective forms of Rubisco had higher rates of CO2 fixation per unit of enzyme than the tobacco control. These transplastomic tobacco lines represent an important step towards improved photosynthesis in plants and will be valuable hosts for future addition of the remaining components of the cyanobacterial CCM, such as inorganic carbon transporters and the ß-carboxysome shell proteins.

Efficient chimeric plant promoters derived from plant infecting viral promoter sequences.

In the present study, we developed a set of three chimeric/hybrid promoters namely FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt incorporating different important domains of Figwort Mosaic Virus sub-genomic transcript promoter (FSgt, -270 to -60), Mirabilis Mosaic Virus sub-genomic transcript promoter (MSgt, -306 to -125) and Peanut Chlorotic Streak Caulimovirus full-length transcript promoter (PFlt-, -353 to +24 and PFlt-UAS, -353 to -49). We demonstrated that these chimeric/hybrid promoters can drive the expression of reporter genes in different plant species including tobacco, Arabidopsis, petunia, tomato and spinach. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 4.2, 1.5 and 1.2 times stronger GUS activities compared to the activity of the CaMV35S promoter, respectively, in tobacco protoplasts. Protoplast-derived recombinant promoter driven GFP showed enhanced accumulation compared to that obtained under the CaMV35S promoter. FSgt-PFlt, PFlt-UAS-2X and MSgt-PFlt promoters showed 3.0, 1.3 and 1.0 times stronger activities than the activity of the CaMV35S² (a modified version of the CaMV35S promoter with double enhancer domain) promoter, respectively, in tobacco (Nicotiana tabacum, var. Samsun NN). Alongside, we observed a fair correlation between recombinant promoter-driven GUS accumulation with the corresponding uidA-mRNA level in transgenic tobacco. Histochemical (X-gluc) staining of whole transgenic seedlings and fluorescence images of ImaGene Green™ treated floral parts expressing the GUS under the control of recombinant promoters also support above findings. Furthermore, we confirmed that these chimeric promoters are inducible in the presence of 150 µM salicylic acid (SA) and abscisic acid (ABA). Taken altogether, we propose that SA/ABA inducible chimeric/recombinant promoters could be used for strong expression of gene(s) of interest in crop plants.

Meiosis in cereal crops: the grasses are back.

A major goal of breeding programs is to increase and manipulate the genetic diversity of crops. The incorporation of beneficial genes from wild relatives into crops is achieved by producing hybrid plants in which meiotic recombination events occur between the two genomes. Furthering our understanding of meiosis in the cereals could enable the manipulation of homolog pairing and recombination, significantly enhancing the efficiency of breeding programs. The main obstacle to the genetic analysis of meiosis in cereal crops has been the complex organization of most cereal genomes, many of which are polyploid. However, the recent sequencing of the rice genome, the use of insertional mutagenesis and reverse genetics approaches has opened the door for the genetic and genomic analysis of cereal meiosis. The goal of this review is to show how these new resources, as well as the use of three-dimensional microscopy, are rapidly providing insights into the mechanisms that control pairing, recombination and segregation of homologous chromosomes during meiosis in four major cereal crops: wheat, rice, maize and rye.