Whole-Genome Duplication Facilitated the Evolution of C4 Photosynthesis in Gynandropsis gynandra.

In higher plants, whole-genome duplication (WGD) is thought to facilitate the evolution of C4 photosynthesis from C3 photosynthesis. To understand this issue, we used new and existing leaf-development transcriptomes to construct two coding sequence databases for C4Gynandropsis gynandra and C3Tarenaya hassleriana, which shared a WGD before their divergence. We compared duplicated genes in the two species and found that the WGD contributed to four aspects of the evolution of C4 photosynthesis in G. gynandra. First, G. gynandra has retained the duplicates of ALAAT (alanine aminotransferase) and GOGAT (glutamine oxoglutarate aminotransferase) for nitrogen recycling to establish a photorespiratory CO2 pump in bundle sheath (BS) cells for increasing photosynthesis efficiency, suggesting that G. gynandra experienced a C3-C4 intermediate stage during the C4 evolution. Second, G. gynandra has retained almost all known vein-development-related paralogous genes derived from the WGD event, likely contributing to the high vein complexity of G. gynandra. Third, the WGD facilitated the evolution of C4 enzyme genes and their recruitment into the C4 pathway. Fourth, several genes encoding photosystem I proteins were derived from the WGD and are upregulated in G. gynandra, likely enabling the NADH dehydrogenase-like complex to produce extra ATPs for the C4 CO2 concentration mechanism. Thus, the WGD apparently played an enabler role in the evolution of C4 photosynthesis in G. gynandra. Importantly, an ALAAT duplicate became highly expressed in BS cells in G. gynandra, facilitating nitrogen recycling and transition to the C4 cycle. This study revealed how WDG may facilitate C4 photosynthesis evolution.

Elevated auxin biosynthesis and transport underlie high vein density in C4 leaves.

High vein density, a distinctive trait of C4 leaves, is central to both C3-to-C4 evolution and conversion of C3 to C4-like crops. We tested the hypothesis that high vein density in C4 leaves is due to elevated auxin biosynthesis and transport in developing leaves. Up-regulation of genes in auxin biosynthesis pathways and higher auxin content were found in developing C4 leaves compared with developing C3 leaves. The same observation held for maize foliar (C4) and husk (C3) leaf primordia. Moreover, auxin content and vein density were increased in loss-of-function mutants of Arabidopsis MYC2, a suppressor of auxin biosynthesis. Treatment with an auxin biosynthesis inhibitor or an auxin transport inhibitor led to much fewer veins in new leaves. Finally, both Arabidopsis thaliana auxin efflux transporter pin1 and influx transporter lax2 mutants showed reduced vein numbers. Thus, development of high leaf vein density requires elevated auxin biosynthesis and transport.

C4 leaf development and evolution.

C4 photosynthesis is more efficient than C3 photosynthesis for two reasons. First, C4 plants have evolved efficient C4 enzymes to suppress wasteful photorespiration and enhance CO2 fixation. Second, C4 leaves have Kranz anatomy in which the veins are surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. The BS and M cells are functionally well differentiated and also well coordinated for rapid assimilation of atmospheric CO2 and transport of photo-assimilates between the two types of cells. Recent comparative transcriptomics of developing M and BS cells in young maize embryonic leaves revealed not only potential regulators of BS and M cell differentiation but also rapid early BS cell differentiation whereas slower, more prolonged M cell differentiation, contrary to the traditional view of a far simpler process of M cell development. Moreover, new upstream regulators of Kranz anatomy development have been identified and a number of gene co-expression modules for early vascular development have been inferred. Also, a candidate gene regulatory network associated with Kranz anatomy and vascular development has been constructed. Additionally, how whole genome duplication (WGD) may facilitate C4 evolution has been studied and the reasons for why the same WGD event led to successful C4 evolution in Gynandropsis gynandra but not in the sister species Tarenaya hassleriana have been proposed. Finally, new future research directions are suggested.

Insights into the regulation of C4 leaf development from comparative transcriptomic analysis.

C4 photosynthesis is more efficient than C3 photosynthesis for two reasons. First, C4 plants have evolved a repertoire of C4 enzymes to enhance CO2 fixation. Second, C4 leaves have Kranz anatomy with a high vein density in which the veins are surrounded by one layer of bundle sheath (BS) cells and one layer of mesophyll (M) cells. The BS and M cells are not only functionally well differentiated, but also well-coordinated for rapid transport of photo-assimilates between the two types of photosynthetic cells. Recent comparative transcriptomic and anatomical analyses of C3 and C4 leaves have revealed early onset of C4-related processes in leaf development, suggesting that delayed mesophyll differentiation contributes to higher C4 vein density, and have identified some candidate regulators for the higher vein density in C4 leaves. Moreover, comparative transcriptomics of maize husk (C3) and foliar leaves (C4) has identified a cohort of candidate regulators of Kranz anatomy development. In addition, there has been major progress in the identification of transcription factor binding sites, greatly increasing our knowledge of gene regulation in plants.