From the archives: Meristem transitions in wheat spike development, detox of reactive carbonyl species in plant metabolism, and transformation of Arabidopsis cells

December 2021: meristem transitions in wheat spike development

Seed number is a major component of yield and a target for crop breeding. In wheat, grains are formed within a spike composed of several spikelets. Once reproductive development starts, the inflorescence meristem within a spike generates the spikelets until a terminal spike is formed. A complex network of transcription factors controls the architecture of spikes and spikelets: for instance, VERNALIZATION1 (VRN1), FRUITFULL (FUL2), and FUL3 (members of the SQUAMOSA clade). Previous analysis showed that the vrn1 ful2 double mutant has defects in spikelet identity specification and inflorescence formation (Li et al., 2019). In 2021, Kun Li, Juan Dibernardi, and colleagues (Li et al., 2021) studied the regulatory network acting downstream of VRN1–FUL2 in controlling the early stages of reproductive development in wheat. The authors conducted transcriptomics analysis on the vrn1 and vrn1 ful2 mutants and found genes in the SHORT VEGETATIVE PHASE (SVP) clade displaying upregulated patterns of expression related to meristem transitions. The vrt2 and svp1 single mutants were shorter and displayed delayed flowering. Remarkably, the vrt2 and svp1 mutants had more spikelets, whereas in the vrt2 svp1 double mutant, the number of spikelets was higher than the sum of the effects of single mutations (see Figure). Further evidence for the involvement of an SVP in the control of spike development came from the overexpression of VRT2 (also known as SVP2), which caused alteration in floret development, linked to a reduction in the expression of VRN1, PISTILLATA 1, AGAMOUS 1, and SEPALLATA genes, which encodes A, B, C, and E-class MADS-box transcription factors, respectively. Finally, the authors tested protein interactions between SQUAMOSA, SEPALLATA, and SVP proteins. This work addressed several layers of regulation controlling spike development in wheat.

Figure.

Effects of vrt2 and svp1 mutations on agronomic traits. A, Plants 80 days after planting. B, Spikes. Adapted from Li et al. (2021), Figure 3.

December 2017: detox of reactive carbonyl species in plant metabolism

A plant’s central metabolism often produces toxic byproducts. For instance, methylglyoxal (MGO) and glyoxal (GO) are mainly produced from the autoxidation of glucose and conversion of triose phosphates. Additionally, some plants accumulate MGO as a response to abiotic stresses. GO and MGO are highly reactive and can modify different cellular macromolecules. Five years ago, Jessica Schmitz and colleagues (Schmitz et al., 2017) characterized the glyoxalase (GLX) system, the main pathway for GO and MGO detoxification in Arabidopsis. The GLX pathway involves two enzymes, GLXI and GLXII. The Arabidopsis genome encodes three GLX1 genes and three GLXII genes. Several splice forms are predicted to be translated and generate isoforms that can be targeted to the cytosol, chloroplasts, or mitochondria. In the vegetative and reproductive stages of Arabidopsis, the authors observed high abundances of transcripts encoding the isoforms GLXI;1_1, GLXI;2_1, GLXI;3_1, and GLXI;3_4, and confirmed the occurrence of all GLXII alternative splice forms. The activity of the GLX enzymes depends on a metal co-factor. Analysis of enzymatic activity indicated that GLXI;1 and GLXI;2 are Ni²⁺ dependent, while GLXI;3 is a Zn²⁺-dependent enzyme. Additional analysis to test metal activation profiles revealed that the GLX system can detoxify not only MGO but also GO and that this depends on the metal co-factor used for catalysis. Enzymatic activity was also tested in vivo using the glo1Δ yeast strain with defects in GLOI, a Zn²⁺-dependent GLXI in yeast. The constitutive expression of GLXI;1, GLXI;2, or GLXI;3 complemented the normal growth of the glo1Δ knockout strain indicating those isoforms are active GLXI enzymes that can detoxify both substrates in vivo. The authors generated fluorescent marker lines to analyze the distribution of all predominant splice forms of GLXII and found a dual targeting to mitochondria and chloroplasts. In summary, this work presented gene expression, enzyme activity, and protein distribution of the GLX enzymes and presented a sub-cellular model for MGO and GO detoxification in Arabidopsis.

December 1997: transformation of Arabidopsis cells

At the end of the last century, plant genetic transformation opened the door for a whole new world of discoveries and experiments. Twenty-five years ago, work by Christoph Forreiter and colleagues (Forreiter et al., 1997) developed a protocol for the genetic transformation of cell cultures that allowed transient experiments. The authors introduced the firefly luciferase (Luc) gene into Arabidopsis cell suspension cultures and performed an analysis on chaperon activity in vivo. The LUC protein is sensitive to temperature changes affecting its activity. In this study, the authors exploited this feature to quantify the activity of chaperones in the protection from heat-induced denaturation and activity recovery. The authors used the stable line of Arabidopsis expressing the Luc gene to generate protoplast and study Luc thermal denaturation. The activity of Luc after heat shock treatments revealed that Luc half-life at 40°C is around 8 min, at 41°C is 5 min, and at 42°C is <3 min. For further analysis, the authors selected 41°C for heat-induced denaturation. The first set of experiments was conducted to study the thermal denaturation of Luc in the presence of chaperones. They selected three heat shock proteins (Hsp): Hsp17.6 from Arabidopsis, Hsp70 from petunia, and Hsp90 from Brassica to test transient expression in the protoplast of the Luc line. Hsp90 and Hsp70 did not have a protective effect; however, Hsp17.6 doubled the half-life at 41°C from 4 to 8–9 min.