Transgenic expression of Rubisco accumulation factor2 and Rubisco subunits increases photosynthesis and growth in maize.

Carbon assimilation by Rubisco is often a limitation to photosynthesis and therefore plant productivity. We have previously shown that transgenic co-expression of the Rubisco large (LS) and small (SS) subunits along with an essential Rubisco accumulation factor, Raf1, leads to faster growth, increased photosynthesis, and enhanced chilling tolerance in maize (Zea mays). Maize also requires Rubisco accumulation factor2 (Raf2) for full accumulation of Rubisco. Here we have analyzed transgenic maize lines with increased expression of Raf2 or Raf2 plus LS and SS. We show that increasing Raf2 expression alone had minor effects on photosynthesis, whereas expressing Raf2 with Rubisco subunits led to increased Rubisco content, more rapid carbon assimilation, and greater plant height, most notably in plants at least 6 weeks of age. The magnitude of the effects was similar to what was observed previously for expression of Raf1 together with Rubisco subunits. Taken together, this suggests that increasing the amount of either assembly factor with Rubisco subunits can independently enhance Rubisco abundance and some aspects of plant performance. These results could also imply either synergy or a degree of functional redundancy for Raf1 and Raf2, the latter of whose precise role in Rubisco assembly is currently unknown.

A linker protein from a red-type pyrenoid phase separates with Rubisco via oligomerizing sticker motifs.

The slow kinetics and poor substrate specificity of the key photosynthetic CO2-fixing enzyme Rubisco have prompted the repeated evolution of Rubisco-containing biomolecular condensates known as pyrenoids in the majority of eukaryotic microalgae. Diatoms dominate marine photosynthesis, but the interactions underlying their pyrenoids are unknown. Here, we identify and characterize the Rubisco linker protein PYCO1 from Phaeodactylum tricornutum. PYCO1 is a tandem repeat protein containing prion-like domains that localizes to the pyrenoid. It undergoes homotypic liquid-liquid phase separation (LLPS) to form condensates that specifically partition diatom Rubisco. Saturation of PYCO1 condensates with Rubisco greatly reduces the mobility of droplet components. Cryo-electron microscopy and mutagenesis data revealed the sticker motifs required for homotypic and heterotypic phase separation. Our data indicate that the PYCO1-Rubisco network is cross-linked by PYCO1 stickers that oligomerize to bind to the small subunits lining the central solvent channel of the Rubisco holoenzyme. A second sticker motif binds to the large subunit. Pyrenoidal Rubisco condensates are highly diverse and tractable models of functional LLPS.

Red Rubiscos and opportunities for engineering green plants.

Nature's vital, but notoriously inefficient, CO2-fixing enzyme Rubisco often limits the growth of photosynthetic organisms including crop species. Form I Rubiscos comprise eight catalytic large subunits and eight auxiliary small subunits and can be classified into two distinct lineages-'red' and 'green'. While red-type Rubiscos (Form IC and ID) are found in rhodophytes, their secondary symbionts, and certain proteobacteria, green-type Rubiscos (Form IA and IB) exist in terrestrial plants, chlorophytes, cyanobacteria, and other proteobacteria. Eukaryotic red-type Rubiscos exhibit desirable kinetic properties, namely high specificity and high catalytic efficiency, with certain isoforms outperforming green-type Rubiscos. However, it is not yet possible to functionally express a high-performing red-type Rubisco in chloroplasts to boost photosynthetic carbon assimilation in green plants. Understanding the molecular and evolutionary basis for divergence between red- and green-type Rubiscos could help us to harness the superior CO2-fixing power of red-type Rubiscos. Here we review our current understanding about red-type Rubisco distribution, biogenesis, and sequence-structure, and present opportunities and challenges for utilizing red-type Rubisco kinetics towards crop improvements.

CO2 -fixing liquid droplets: Towards a dissection of the microalgal pyrenoid.

CO2 enters the biosphere via the slow, oxygen-sensitive carboxylase, Rubisco. To compensate, most microalgae saturate Rubisco with its substrate gas through a carbon dioxide concentrating mechanism. This strategy frequently involves compartmentalization of the enzyme in the pyrenoid, a non-membrane enclosed compartment of the chloroplast stroma. Recently, tremendous advances have been achieved concerning the structure, physical properties, composition and in vitro reconstitution of the pyrenoid matrix from the green alga Chlamydomonas reinhardtii. The discovery of the intrinsically disordered multivalent Rubisco linker protein EPYC1 provided a biochemical framework to explain the subsequent finding that the pyrenoid resembles a liquid droplet in vivo. Reconstitution of the corresponding liquid-liquid phase separation using pure Rubisco and EPYC1 allowed a detailed characterization of this process. Finally, a large high-quality dataset of pyrenoidal protein-protein interactions inclusive of spatial information provides ample substrate for rapid further functional dissection of the pyrenoid. Integrating and extending recent advances will inform synthetic biology efforts towards enhancing plant photosynthesis as well as contribute a versatile model towards experimentally dissecting the biochemistry of enzyme-containing membraneless organelles.

Characterization of Silver Nanoparticles Internalized by Arabidopsis Plants Using Single Particle ICP-MS Analysis.

Plants act as a crucial interface between humans and their environment. The wide use of nanoparticles (NPs) has raised great concerns about their potential impacts on crop health and food safety, leading to an emerging research theme about the interaction between plants and NPs. However, up to this day even the basic issues concerning the eventual fate and characteristics of NPs after internalization are not clearly delineated due to the lack of a well-established technique for the quantitative analysis of NPs in plant tissues. We endeavored to combine a quantitative approach for NP analysis in plant tissues with TEM to localize the NPs. After using an enzymatic digestion to release the NPs from plant matrices, single particle-inductively coupled plasma-mass spectrometry (SP-ICP-MS) is employed to determine the size distribution of silver nanoparticles (Ag NPs) in tissues of the model plant Arabidopsis thaliana after exposure to 10 nm Ag NPs. Our results show that Macerozyme R-10 treatment can release Ag NPs from Arabidopsis plants without changing the size of Ag NPs. The characteristics of Ag NPs obtained by SP-ICP-MS in both roots and shoots are in agreement with our transmission electron micrographs, demonstrating that the combination of an enzymatic digestion procedure with SP-ICP-MS is a powerful technique for quantitative determination of NPs in plant tissues. Our data reveal that Ag NPs tend to accumulate predominantly in the apoplast of root tissues whereby a minor portion is transported to shoot tissues. Furthermore, the fact that the measured size distribution of Ag NPs in plant tissue is centered at around 20.70 nm, which is larger than the initial 12.84 nm NP diameter, strongly implies that many internalized Ag NPs do not exist as intact individual particles anymore but are aggregated and/or biotransformed in the plant instead.