To study the regulatory and functional differentiation between the mesophyll (M) and bundle sheath (BS) cells of maize (Zea mays), we isolated large quantities of highly homogeneous M and BS cells from newly matured second leaves for transcriptome profiling by RNA sequencing. A total of 52,421 annotated genes with at least one read were found in the two transcriptomes. Defining a gene with more than one read per kilobase per million mapped reads as expressed, we identified 18,482 expressed genes; 14,972 were expressed in M cells, including 53 M-enriched transcription factor (TF) genes, whereas 17,269 were expressed in BS cells, including 214 BS-enriched TF genes. Interestingly, many TF gene families show a conspicuous BS preference in expression. Pathway analyses reveal differentiation between the two cell types in various functional categories, with the M cells playing more important roles in light reaction, protein synthesis and folding, tetrapyrrole synthesis, and RNA binding, while the BS cells specialize in transport, signaling, protein degradation and posttranslational modification, major carbon, hydrogen, and oxygen metabolism, cell division and organization, and development. Genes coding for several transporters involved in the shuttle of C 4 metabolites and BS cell wall development have been identified, to our knowledge, for the first time. This comprehensive data set will be useful for studying M/BS differentiation in regulation and function.C 4 plants, with few exceptions, require the coordination of the mesophyll (M) and bundle sheath (BS) cells, arranged in a wreath structure called Kranz leaf anatomy (Hatch and Agostino, 1992), to confer high rates of photosynthesis. The initial carboxylation phase of the C 4 pathway takes place in the M cells, while the decarboxylation phase is restricted to the BS cells. The high photosynthetic capacity of C 4 plants implies a massive efflux of C 4 -related metabolites between M and BS cells and between the cytosol and organelles in each cell type (Weber and von Caemmerer, 2010).Although research in the past few decades has greatly increased our understanding of the biochemical reactions and the enzymes involved in the C 4 pathway of photosynthesis, little is known about the specific genes involved in the development of the Kranz leaf anatomy, the C 4 biochemical pathway, and the underlying regulatory mechanisms for the high-level expression of C 4 -specific genes in a cell-, organ-, or development-specific manner. With the advancement in genomics, the genomic sequences of several C 3 and C 4 model plants have become available. These advances have allowed in-depth comparative proteomic and transcriptomic analyses of the whole leaves of typical C 3 and C 4 plants and their closely related C 3 -C 4 intermediate species of Cleome and Flaveria . These comparative studies allow deduction on how many genes are required to make a C 4 plant and possibly on how they may have been regulated at the genetic level. In addition, attempts have been made recently to characterize...

Time-series transcriptomes of a biological process obtained under different conditions are useful for identifying the regulators of the process and their regulatory networks. However, such data are 3D (gene expression, time, and condition), and there is currently no method that can deal with their full complexity. Here, we developed a method that avoids time-point alignment and normalization between conditions. We applied it to analyze time-series transcriptomes of developing maize leaves under light–dark cycles and under total darkness and obtained eight time-ordered gene coexpression networks (TO-GCNs), which can be used to predict upstream regulators of any genes in the GCNs. One of the eight TO-GCNs is light-independent and likely includes all genes involved in the development of Kranz anatomy, which is a structure crucial for the high efficiency of photosynthesis in C4 plants. Using this TO-GCN, we predicted and experimentally validated a regulatory cascade upstream of SHORTROOT1, a key Kranz anatomy regulator. Moreover, we applied the method to compare transcriptomes from maize and rice leaf segments and identified regulators of maize C4 enzyme genes and RUBISCO SMALL SUBUNIT2. Our study provides not only a powerful method but also novel insights into the regulatory networks underlying Kranz anatomy development and C4 photosynthesis.

Cerebral arterial remodeling is one of the critical factors in the occurrence of postspaceflight orthostatic intolerance. We hypothesize that large-conductance calcium-activated K(+) (BK(Ca)) channels in vascular smooth muscle cells (VSMCs) may play an important role in regulating cerebrovascular adaptation during microgravity exposure. The aim of this work was to investigate whether activation of BK(Ca) channels is involved in regulation of apoptotic remodeling of cerebral arteries in simulated microgravity rats. In animal studies, Sprague-Dawley rats were subjected to 1-wk hindlimb unweighting to simulate microgravity. Alterations of BK(Ca) channels in cerebral VSMCs were investigated by patch clamp and Western blotting; apoptosis was assessed by electron microscopy and terminal deoxynucleotidyltransferase-mediated dUTP-biotin nick-end labeling (TUNEL). To evaluate the correlation of BK(Ca) channel and apoptosis, channel protein and cell nucleus were double-stained. In cell studies, hSloalpha+beta1 channel was coexpressed into human embryonic kidney 293 (HEK293) cells to observe the effects of BK(Ca) channels on apoptosis. In rats, enhanced activities and expression of BK(Ca) channels were found to be correlated with increased apoptosis in cerebral VSMCs after simulated microgravity. In transfected HEK293 cells, activation of cloned BK(Ca) channel induced apoptosis, whereas inhibition of cloned BK(Ca) channel decreased apoptosis. In conclusion, activation of BK(Ca) channels is associated with increased apoptosis in cerebral VSMCs of simulated microgravity rats.