Variations of photosynthetic structures in different tissues or cells are in coordination with changes in various aspects, e.g. physiology, biochemistry, gene expression, etc. Most C 4 plant species undergo developmental enhancement of the photosynthetic system, which may present different modes of changes between anatomy and physiology/biochemistry. In the current study, we investigated a Kranz-type C 4 species Salsola ferganica with the progressive development of photosynthetic (PS) structure, performance of PS physiology, induction of PS enzymes, and transcriptional and translational regulation of PS genes, results revealed that S. ferganica presented C 3 type anatomy in cotyledons but C 4 type in leaves (C 3 /L 4), with the C 4 system separation of initial carbon fixation in the palisade mesophyll (M) cells and the following incorporation into triosephosphates and sugars in the bundle sheath (BS) cells, respectively. The BS cells continuously surrounded the vascular bundles and water storage cells in leaf anatomic structure. Compared to the single-cell C 4 species Suaeda aralocaspica, S. ferganica exhibited similar developmental enhancement of C 4 syndrome temporally and spatially in anatomic structures, enzyme activities, and gene expression, which suggests that completion of differentiation of the photosynthetic system is necessary for a C 4 assimilation pathway. Besides, S. ferganica also displayed some different characteristics compared to S. aralocaspica in photosynthetic physiology, e.g. a more flexible d 13 C value, much lower phosphoenolpyruvate carboxylase (PEPC) activity, and an insensitive response to stimuli, etc., which were not typical C 4 characteristics. We speculate that this may suggest a different status of these two species in the evolutionary process of the photosynthesis pathway. Our findings will contribute to further understanding of the diversity of photosynthesis systems in Kranz-type C 4 species and the Salsola genus.

Seed heteromorphism (SH) is an adaptive strategy towards adversity in many halophytes. However, the underlying mechanisms and ecological significance of SH have not been deeply explored. Using Suaeda aralocaspica, a typical C4 annual halophyte without Kranz anatomy, we studied seed morphology, differentiation of morphs and fruit-setting patterns, and correlated these traits with germination responses, seed characteristics and heteromorphic seed ratio. To elucidate the genetic basis of SH, we analyzed correlated patterns of gene-expression for seed development related genes as well. We observed that S. aralocaspica produced three types of seed morph: brown, large black and small black with differences in color, size, mass and germination behavior; the latter two were further distinguished by their origin in female or bisexual flowers respectively. Further analysis revealed that SH was associated with genetic aspects including seed positioning, seed coat differentiation and seed developmental gene expression, while variations in SH may be associated with environmental conditions, e.g. annual precipitation, temperature, daylight, and their monthly distribution in different calendar years. SH and its variations in S. aralocaspica show multi-level regulation of the bet-hedging strategy that influences phenotypic plasticity, which is a consequence of internal genetic and external environmental factor interaction. Our findings contribute to the understanding of SH as a potential adaptive trait of desert plant species.

Phosphoenolpyruvate carboxylase (PEPC) plays pivotal roles in the carbon fixation of photosynthesis and a variety of metabolic and stress pathways. Suaeda aralocaspica belongs to a single-cellular C4 species and carries out a photosynthetic pathway in an unusually elongated chlorenchyma cell, which is expected to have PEPCs with different characteristics. To identify the different isoforms of PEPC genes in S. aralocaspica and comparatively analyze their expression and regulation patterns as well as the biochemical and enzymatic properties in this study, we characterized a bacterial-type PEPC (BTPC; SaPEPC-4) in addition to the two plant-type PEPCs (PTPCs; SaPEPC-1 and SaPEPC-2) using a genome-wide identification. SaPEPC-4 presented a lower expression level in all test combinations with an unknown function; two SaPTPCs showed distinct subcellular localizations and different spatiotemporal expression patterns but positively responded to abiotic stresses. Compared to SaPEPC-2, the expression of SaPEPC-1 specifically in chlorenchyma cell tissues was much more active with the progression of development and under various stresses, particularly sensitive to light, implying the involvement of SaPEPC-1 in a C4 photosynthetic pathway. In contrast, SaPEPC-2 was more like a non-photosynthetic PEPC. The expression trends of two SaPTPCs in response to light, development, and abiotic stresses were also matched with the changes in PEPC activity in vivo (native) or in vitro (recombinant), and the biochemical properties of the two recombinant SaPTPCs were similar in response to various effectors while the catalytic efficiency, substrate affinity, and enzyme activity of SaPEPC-2 were higher than that of SaPEPC-1 in vitro. All the different properties between these two SaPTPCs might be involved in transcriptional (e.g., specific cis-elements), posttranscriptional [e.g., 5′-untranslated region (5′-UTR) secondary structure], or translational (e.g., PEPC phosphorylation/dephosphorylation) regulatory events. The comparative studies on the different isoforms of the PEPC gene family in S. aralocaspica may help to decipher their exact role in C4 photosynthesis, plant growth/development, and stress resistance.