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...

The genus Flaveria shows evidence of evolution in the mechanism of photosynthesis as its 21 species include C3, C3-C4, C4-like, and C4 plants. In this study, several physiological and biochemical parameters of photosynthesis and photorespiration were measured in 18 Flaveria species representing all the photosynthetic types. The 10 species classified as C3-C4 intermediates showed an inverse continuum in level of photorespiration and development of the C4 syndrome. This ranges from F. sonorensis with relatively high apparent photorespiration and lacking C4 photosynthesis to F. Among the intermediates, the photosynthetic C02 compensation points at 300C and 1150 micromoles quanta per square meter per second varied from 9 to 29 microbars. The values for the three C4-like species varied from 3 to 6 microbars, similar to those measured for the C4 species. The activities of the photorespiratory enzymes glycolate oxidase, hydroxypynrvate reductase, and serine hydroxymethyltransferase decreased progressively from C3 to C3-C4 to C4-like and C4 species. On tlh other hand, most intermediates had higher levels of phosphenaIhuvate carboxylase and NADP-malic enzyme than C3 species, but generally lower activities compared to C4-like and C4 species. The levels of these C4 enzymes are correlated with the degree of C4 photosynthesis, based on the initial products of photosynthesis. Another indication of development of the C4 syndrome in C3-C4 Flaveria species was their intermediate chlorophyll a/b rafos. The chlorophyll a/b ratios of the various Flaveria species are highly correlated with the degree of C4 photosynthesis suggesting that the photochemical machinery is progressively altered during evolution in order to meet the specific energy requirements for operating the C4 pathway. In the progression from C3 to C4 species in Flaveria, the CO2 compensation point decreased more rapidly than did the decrease in 02 inhibition of photosynthesis or the increase in the degree of C4 photosynthesis. These results suggest that the reduction in photorespiration during evolution occurred initially by refixation of photorespired CO2 and prior to substantive reduction in 02 inhibition and development of the C4 syndrome. However, further reduction in 03 inhibition in some intermediates and C4-like species is considered primarily due to the development of the C4 syndrome. Thus, the evolution of C3-C4 intermediate photosynthesis likely occurred in response to environmental conditions which limit the intercellular CO2 concentration first via refixation of photorespired C02, followed by development of the C4 syndrome.

Male sterility plays an important role in F1 hybrid seed production. We identified a male-sterile rice (Oryza sativa) mutant with impaired pollen development and a single T-DNA insertion in the transcription factor gene bHLH142. Knockout mutants of bHLH142 exhibited retarded meiosis and defects in tapetal programmed cell death. RT-PCR and in situ hybridization analyses showed that bHLH142 is specifically expressed in the anther, in the tapetum, and in meiocytes during early meiosis. Three basic helix-loop-helix transcription factors, UDT1 (bHLH164), TDR1 (bHLH5), and EAT1/DTD1 (bHLH141) are known to function in rice pollen development. bHLH142 acts downstream of UDT1 and GAMYB but upstream of TDR1 and EAT1 in pollen development. In vivo and in vitro assays demonstrated that bHLH142 and TDR1 proteins interact. Transient promoter assays demonstrated that regulation of the EAT1 promoter requires bHLH142 and TDR1. Consistent with these results, 3D protein structure modeling predicted that bHLH142 and TDR1 form a heterodimer to bind to the EAT1 promoter. EAT1 positively regulates the expression of AP37 and AP25, which induce tapetal programmed cell death. Thus, in this study, we identified bHLH142 as having a pivotal role in tapetal programmed cell death and pollen development.

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Using an Agrobacterium-mediated transformation system, we have introduced the intact gene of maize phosphoenolpyruvate carboxylase (PEPC), which catalyzes the initial fixation of atmospheric CO2 in C4 plants into the C3 crop rice. Most transgenic rice plants showed high-level expression of the maize gene; the activities of PEPC in leaves of some transgenic plants were two- to threefold higher than those in maize, and the enzyme accounted for up to 12% of the total leaf soluble protein. RNA gel blot and Southern blot analyses showed that the level of expression of the maize PEPC in transgenic rice plants correlated with the amount of transcript and the copy number of the inserted maize gene. Physiologically, the transgenic plants exhibited reduced O2 inhibition of photosynthesis and photosynthetic rates comparable to those of untransformed plants. The results demonstrate a successful strategy for installing the key biochemical component of the C4 pathway of photosynthesis in C3 plants.

Although the enucleate conducting cells of the phloem are incapable of protein synthesis, phloem exudates characteristically contain low concentrations of soluble proteins. The role of these proteins and their movement into and out of the sieve tubes poses important questions for phloem physiology and for cell-to-cell protein movement via plasmodesmata. Because mature sieve elements lack both a nucleus and ribosomes (4), they are incapable of protein synthesis. Clearly, the ongoing presence of proteins in the translocation stream requires their continual replacement by movement from adjacent nucleate cells. Companion cells are the most likely origin of such proteins and characteristically possess an active-appearing cytoplasm, including abundant ribosomes. Recently, Nakamura et al. (16) (17) showed that even 'structural' P-proteins are involved in rapid turnover and presented microautoradiographic evidence for their synthesis in companion cells.The occurrence of protein turnover in sieve tubes raises a number of intriguing and physiologically significant questions. Movement of proteins into and out of the sieve tube presumably occurs via plasmodesmata, which, except for pathological conditions, appear to provide passageways too small for intercellular protein movement (21). Although their functions are unknown, the proteins presumably have some role in source-sink and/or sieve tube-companion cell relations. As Raven (20) recently emphasized, the striking longevity of sieve elements as enucleate cells poses ongoing maintenance problems that almost certainly require intercellular protein transport. Finally, the synthesis and movement of phloem proteins in healthy plants may provide insight into the replication and movement of phloem-limited viruses and Mycoplasma-like organisms.The following experiments were undertaken to investigate some of the overall characteristics of soluble sieve tube proteins, especially their number and variability along the transport pathway, and their pattems of synthesis, transport, and turnover. MATERIALS AND METHODS Plant MaterialWheat plants (Triticum aestivum L. cv SUN 9E) were grown in a growth chamber as described previously (9). Experiments were performed with plants in the middle portion of the grain-filling stage (approximately 15-25 d after anthesis).

The potential for C4 photosynthesis was investigated in five C3-C4 intermediate species, one C3 species, and one C4 species in the genus Flaveria, using (14)CO2 pulse-(12)CO2 chase techniques and quantum-yield measurements. All five intermediate species were capable of incorporating (14)CO2 into the C4 acids malate and aspartate, following an 8-s pulse. The proportion of (14)C label in these C4 products ranged from 50-55% to 20-26% in the C3-C4 intermediates F. floridana Johnston and F. linearis Lag. respectively. All of the intermediate species incorporated as much, or more, (14)CO2 into aspartate as into malate. Generally, about 5-15% of the initial label in these species appeared as other organic acids. There was variation in the capacity for C4 photosynthesis among the intermediate species based on the apparent rate of conversion of (14)C label from the C4 cycle to the C3 cycle. In intermediate species such as F. pubescens Rydb., F. ramosissima Klatt., and F. floridana we observed a substantial decrease in label of C4-cycle products and an increase in percentage label in C3-cycle products during chase periods with (12)CO2, although the rate of change was slower than in the C4 species, F. palmeri. In these C3-C4 intermediates both sucrose and fumarate were predominant products after a 20-min chase period. In the C3-C4 intermediates, F. anomala Robinson and f. linearis we observed no significant decrease in the label of C4-cycle products during a 3-min chase period and a slow turnover during a 20-min chase, indicating a lower level of functional integration between the C4 and C3 cycles in these species, relative to the other intermediates. Although F. cronquistii Powell was previously identified as a C3 species, 7-18% of the initial label was in malate+aspartate. However, only 40-50% of this label was in the C-4 position, indicating C4-acid formation as secondary products of photosynthesis in F. cronquistii. In 21% O2, the absorbed quantum yields for CO2 uptake (in mol CO2·[mol quanta](-1)) averaged 0.053 in F. cronquistii (C3), 0.051 in F. trinervia (Spreng.) Mohr (C4), 0.052 in F. ramosissima (C3-C4), 0.051 in F. anomala (C3-C4), 0.050 in F. linearis (C3-C4), 0.046 in F. floridana (C3-C4), and 0.044 in F. pubescens (C3-C4). In 2% O2 an enhancement of the quantum yield was observed in all of the C3-C4 intermediate species, ranging from 21% in F. ramosissima to 43% in F. pubescens. In all intermediates the quantum yields in 2% O2 were intermediate in value to the C3 and C4 species, indicating a co-function of the C3 and C4 cycles in CO2 assimilation. The low quantum-yield values for F. pubescens and F. floridana in 21% O2 presumably reflect an ineffcient transfer of carbon from the C4 to the C3 cycle. The response of the quantum yield to four increasing O2 concentrations (2-35%) showed lower levels of O2 inhibition in the C3-C4 intermediate F. ramosissima, relative to the C3 species. This indicates that the co-function of the C3 and C4 cycles in this intermediate species leads to an increased CO2 concentration ...

BackgroundCytokinins are plant-specific hormones that affect plant growth and development. The endogenous level of cytokinins in plant cells is regulated in part by irreversible degradation via cytokinin oxidase/dehydrogenase (CKX). Among the 11 rice CKXs, CKX2 has been implicated in regulation of rice grain yield.ResultsTo specifically down-regulate OsCKX2 expression, we have chosen two conserved glycosylation regions of OsCKX2 for designing artificial short hairpin RNA interference genes (shRNA-CX3 and -CX5, representing the 5′ and 3′ glycosylation region sequences, respectively) for transformation by the Agrobacterium-mediated method. For each construct, 5 independent transgenic lines were obtained for detailed analysis. Southern blot analysis confirmed the integration of the shRNA genes into the rice genome, and quantitative real time RT-PCR and northern blot analyses showed reduced OsCKX2 expression in the young stem of transgenic rice at varying degrees. However, the expression of other rice CKX genes, such as CKX1 and CKX3, in these transgenic lines was not altered. Transgenic rice plants grown in the greenhouse were greener and more vigorous with delayed senescence, compared to the wild type. In field experiments, both CX3 and CX5 transgenic rice plants produced more tillers (27–81 %) and grains (24–67 %) per plant and had a heavier 1000 grain weight (5–15 %) than the wild type. The increases in grain yield were highly correlated with increased tiller numbers. Consistently, insertional activation of OsCKX2 led to increased expression of CKX2 and reduced tiller number and growth in a gene-dosage dependant manner.ConclusionsTaken together, these results demonstrate that specific suppression of OsCKX2 expression through shRNA-mediated gene silencing leads to enhanced growth and productivity in rice by increasing tiller number and grain weight.Electronic supplementary materialThe online version of this article (doi:10.1186/s12284-015-0070-5) contains supplementary material, which is available to authorized users.