Morphological change and genome-wide transcript analysis of Haloxylon ammodendron leaf development reveals morphological characteristics and genes associated with the different C3 and C4 photosynthetic metabolic pathways

C4 photosynthesis outperforms C3 photosynthesis in natural ecosystems by maintaining a high photosynthetic rate and affording higher water-use and nitrogen-use efficiencies. C4 plants can survive in environments with poor living conditions, such as high temperatures and arid regions, and will be crucial to ecological and agricultural security in the face of global climate change in the future. However, the genetic architecture of C4 photosynthesis remains largely unclear, especially the genetic regulation of C4 Kranz anatomy. Haloxylon ammodendron is an important afforestation tree species and a valuable C4 wood plant in the desert region. The unique characteristic of H. ammodendron is that, during the seedling stage, it utilizes C3 photosynthesis, while in mature assimilating shoots (maAS), it switches to the C4 pathway. This makes an exceptional opportunity for studying the development of the C4 Kranz anatomy and metabolic pathways within individual plants (identical genome). To provide broader insight into the regulation of Kranz anatomy and non-Kranz leaves of the C4 plant H. ammodendron, carbon isotope values, anatomical sections and transcriptome analyses were used to better understand the molecular and cellular processes related to the development of C4 Kranz anatomy. This study revealed that H. ammodendron conducts C3 in the cotyledon before it switches to C4 in AS. However, the switching requires a developmental process. Stable carbon isotope discrimination measurements on three different developmental stages showed that young AS have a C3-like delta 13C even though C4 Kranz anatomy is found, which is inconsistent with the anatomical findings. A C4-like delta 13C can be measured in AS until they are mature. The expression analysis of C4 key genes also showed that the maAS exhibited higher expression than the young AS. In addition, many genes that may be related to the development of Kranz anatomy were screened. Comparison of gene expression patterns with respect to anatomy during leaf ontogeny provided insight into the genetic features of Kranz anatomy. This study helps with our understanding of the development of Kranz anatomy and provides future directions for studies on key C4 regulatory genes.