The evolution of stomatal traits along the trajectory toward C4 photosynthesis.

C4 photosynthesis optimizes plant carbon and water relations, allowing high photosynthetic rates with low stomatal conductance. Stomata have long been considered a part of the C4 syndrome. However, it remains unclear how stomatal traits evolved along the path from C3 to C4. Here, we examined stomata in the Flaveria genus, a model used for C4 evolutionary study. Comparative, transgenic, and semi-in vitro experiments were performed to study the molecular basis that underlies the changes of stomatal traits in C4 evolution. The evolution from C3 to C4 species is accompanied by a gradual rather than an abrupt change in stomatal traits. The initial change appears near the Type I intermediate stage. Co-evolution of the photosynthetic pathway and stomatal traits is supported. On the road to C4, stomata tend to be fewer in number but larger in size and stomatal density dominates changes in anatomical maximum stomatal conductance (gsmax). Reduction of FSTOMAGEN expression underlies decreased gsmax in Flaveria and likely occurs in other C4 lineages. Decreased gsmax contributes to the increase in intrinsic water-use efficiency in C4 evolution. This work highlights the stomatal traits in the current C4 evolutionary model. Our study provides insights into the pattern, mechanism, and role of stomatal evolution along the road toward C4.

EXPLICIT-Kinase: A gene expression predictor for dissecting the functions of the Arabidopsis kinome.

Protein kinases regulate virtually all cellular processes, but it remains challenging to determine the functions of all protein kinases, collectively called the "kinome", in any species. We developed a computational approach called EXPLICIT-Kinase to predict the functions of the Arabidopsis kinome. Because the activities of many kinases can be regulated transcriptionally, their gene expression patterns provide clues to their functions. A universal gene expression predictor for Arabidopsis was constructed to predict the expression of 30,172 non-kinase genes based on the expression of 994 kinases. The model reconstituted highly accurate transcriptomes for diverse Arabidopsis samples. It identified the significant kinases as predictor kinases for predicting the expression of Arabidopsis genes and pathways. Strikingly, these predictor kinases were often regulators of related pathways, as exemplified by those involved in cytokinesis, tissue development, and stress responses. Comparative analyses revealed that portions of these predictor kinases are shared and conserved between Arabidopsis and maize. As an example, we identified a conserved predictor kinase, RAF6, from a stomatal movement module. We verified that RAF6 regulates stomatal closure. It can directly interact with SLAC1, a key anion channel for stomatal closure, and modulate its channel activity. Our approach enables a systematic dissection of the functions of the Arabidopsis kinome.

Rewiring of primary metabolism for ammonium recycling under short-term low CO2 treatment - its implication for C4 evolution.

The dramatic decrease in atmospheric CO2 concentration during Oligocene was proposed as directly linked to C4 evolution. However, it remains unclear how the decreased CO2 concentration directly facilitate C4 evolution, besides its role as a selection pressure. We conducted a systematic transcriptomics and metabolomics analysis under short-term low CO2 condition and found that Arabidopsis grown under this condition showed 1) increased expression of most genes encoding C4-related enzymes and transporters; 2) increased expression of genes involved in photorespiration and pathways related to carbon skeleton generation for ammonium refixation; 3) increased expression of genes directly involved in ammonium refixation. Furthermore, we found that in vitro treatment of leaves with NH4 + induced a similar pattern of changes in C4 related genes and genes involved in ammonium refixation. These data support the view that Arabidopsis grown under short-term low CO2 conditions rewired its metabolism to supply carbon skeleton for ammonium recycling, during which process the expression of C4 genes were up-regulated as a result of a hitchhiking process. This study provides new insights into the adaptation of the C3 model plant Arabidopsis under low CO2 conditions and suggests that low CO2 can facilitate the evolution of C4 photosynthesis beyond the commonly assumed role of being a selection pressure.