SUMMARYMultiple small molecule hormones contribute to growth promotion or restriction in plants. Brassinosteroids (BRs), acting specifically in the epidermis, can both drive and restrict shoot growth. However, our knowledge of how BRs affect meristem size is scant. Here, we study the root meristem and show that BRs are required to maintain normal cell cycle activity and cell expansion. These two processes ensure the coherent gradient of cell progression, from the apical to the basal meristem. In addition, BR activity in the meristem is not accompanied by changes in the expression level of the auxin efflux carriers PIN1, PIN3 and PIN7, which are known to control the extent of mitotic activity and differentiation. We further demonstrate that BR signaling in the root epidermis and not in the inner endodermis, quiescent center (QC) cells or stele cell files is sufficient to control root meristem size. Interestingly, expression of the QC and the stele-enriched MADS-BOX gene AGL42 can be modulated by BRI1 activity solely in the epidermis. The signal from the epidermis is probably transmitted by a different component than BES1 and BZR1 transcription factors, as their direct targets, such as DWF4 and BRox2, are regulated in the same cells that express BRI1. Taken together, our study provides novel insights into the role of BRs in controlling meristem size.

CesA genes are believed to encode the catalytic subunit of cellulose synthase. Identification of nine distinct CesA cDNAs from maize (Zea mays) has allowed us to initiate comparative studies with homologs from Arabidopsis and other plant species. Mapping studies show that closely related CesA genes are not clustered but are found at different chromosomal locations in both Arabidopsis and maize. Furthermore, sequence comparisons among the CesA-deduced proteins show that these cluster in groups wherein orthologs are often more similar than paralogs, indicating that different subclasses evolved prior to the divergence of the monocot and dicot lineages. Studies using reverse transcriptase polymerase chain reaction with gene-specific primers for six of the nine maize genes indicate that all genes are expressed to at least some level in all of the organs examined. However, when expression patterns for a few selected genes from maize and Arabidopsis were analyzed in more detail, they were found to be expressed in unique cell types engaged in either primary or secondary wall synthesis. These studies also indicate that amino acid sequence comparisons, at least in some cases, may have value for prediction of such patterns of gene expression. Such analyses begin to provide insights useful for future genetic engineering of cellulose deposition, in that identification of close orthologs across species may prove useful for prediction of patterns of gene expression and may also aid in prediction of mutant combinations that may be necessary to generate severe phenotypes.Evidence is accumulating to support the notion that some, if not all, of the members of the family of CesA genes in plants encode a glycosyltranferase that plays a key role in the process of cellulose synthesis (for recent reviews, see Brown et al., 1997; Kawagoe and Delmer, 1997, 1998; Delmer, 1999). The deduced proteins from members of this gene family are characterized by the presence of domains that share significant sequence homology with other family 2 glycosyltransferases that are characterized by having conserved motifs surrounding three conserved D residues and a QXXRW motif downstream of D 3 (Campbell et al., 1997). Recent crystallographic evidence supports a model in which the three D residues, in conjunction with a divalent cation, are involved in binding of the UDP-sugar substrate and in catalysis of glycosyltransfer (Charnock and Davies, 1999). In the deduced proteins encoded by most family 2 glycosyltransferases, the domains containing these conserved D residues are consecutive, but the predicted proteins in plants contain a plant-specific conserved and a hypervariable (HVR-2) domain that separate the domains containing these conserved residues. A conserved, extended N-terminal region containing two zinc fingers resembling LIM/Ring domains (Kawagoe and Delmer, 1997) followed by the HVR-1 region also characterizes the plant CesA proteins. Many of these glycosyltransferases, including the plant and bacterial CesA proteins, are predicted to be ancho...

For many years, mRNA abundance has been used as the surrogate measure of gene expression in biological systems. However, recent genome-scale analyses in both bacteria and eukaryotes have revealed that mRNA levels correlate with steady-state protein abundance for only 50-70% of genes, indicating that translation and post-translation processes also play important roles in determining gene expression. What is not yet clear is whether dynamic processes such as cell cycle progression, differentiation, or response to environmental changes change the relationship between mRNA and protein abundance. Here, we describe a systems approach to interrogate promastigote-to-amastigote differentiation in the obligatory intracellular parasitic protozoan Leishmania donovani. Our results indicate that regulation of mRNA levels plays a major role early in the differentiation process, while translation and post-translational regulation are more important in the latter part. In addition, it appears that the differentiation signal causes a transient global increase in the rate of protein synthesis, which is subsequently down-regulated by phosphorylation of α-subunit of translation initiation factor 2. Thus, Leishmania dynamically changes the relationship between mRNA and protein abundance as it adapts to new environmental circumstances. It is likely that similar mechanisms play a more important role than previously recognized in regulation of gene expression in other organisms.

The mechanisms ensuring balanced growth remain a critical question in developmental biology. In plants, this balance relies on spatiotemporal integration of hormonal signaling pathways, but the understanding of the precise contribution of each hormone is just beginning to take form. Brassinosteroid (BR) hormone is shown here to have opposing effects on root meristem size, depending on its site of action. BR is demonstrated to both delay and promote onset of stem cell daughter differentiation, when acting in the outer tissue of the root meristem, the epidermis, and the innermost tissue, the stele, respectively. To understand the molecular basis of this phenomenon, a comprehensive spatiotemporal translatome mapping of Arabidopsis roots was performed. Analyses of wild type and mutants featuring different distributions of BR revealed autonomous, tissuespecific gene responses to BR, implying its contrasting tissue-dependent impact on growth. BR-induced genes were primarily detected in epidermal cells of the basal meristem zone and were enriched by auxinrelated genes. In contrast, repressed BR genes prevailed in the stele of the apical meristem zone. Furthermore, auxin was found to mediate the growth-promoting impact of BR signaling originating in the epidermis, whereas BR signaling in the stele buffered this effect. We propose that context-specific BR activity and responses are oppositely interpreted at the organ level, ensuring coherent growth.

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Leishmania donovani is an intracellular protozoan parasite that causes kala-azar in humans. During infection the extracellular insect forms (promastigotes) undergo rapid differentiation to intracellular amastigotes that proliferates in phagolysosomes of mammalian macrophages. We used microarraybased expression profiling to investigate the time-course of changes in RNA abundance during promastigote-to-amastigote differentiation in a host-free system that mimics this process. These studies revealed that several hundred genes underwent an ordered progression of transient or permanent up-and down-regulation during differentiation. Genes that were permanently upregulated in amastigotes were enriched for transporters and surface proteins, but under-represented in genes involved in protein and other metabolism. Most of these changes occurred late in the differentiation process, when morphological differentiation was essentially complete. Downregulated genes were over-represented in those involved in cell motility, growth and/or maintenance, and these changes generally occurred earlier in the process. Genes that were transiently up-or downregulated during differentiation included those encoding heat shock proteins, ubiquitin hydrolases, RNA binding proteins, protein kinases, a protein phosphatase, and a histone deacetylase. These results suggest that changes in mRNA abundance may be important in signal transduction, as well as protein and mRNA turnover, during differentiation. In addition to these mRNA changes, other transcripts including one or more rRNAs and snoRNAs, and non-coding RNAs from several telomeres, also showed substantial changes in abundance during the differentiation process. This paper provides the first genome-scale quantitative analysis of gene expression during the transition from promastigotes to amastigotes and demonstrates the utility of the host-free differentiation system.

SummaryWe describe the first functional and molecular characterization of an amino acid permease (LdAAP3) from the human parasitic protozoan Leishmania donovani , the causative agent of visceral leishmaniasis in humans. This permease contains 480 amino acids with 11 predicted trans-membrane domains. Expressing LdAAP3 in Saccharomyces cerevisiae mutants revealed that LdAAP3 codes for a highaffinity arginine transporter ( K m 1.9 m M). LdAAP3 is highly specific for arginine as its transport was not inhibited by other amino acids or arginine-related compounds. Using green fluorescence protein (GFP) fused to the N-terminus of LdAAP3, this transporter was localized to the surface membrane of promastigotes. The GFP-LdAAP3 chimera mediated a threefold increase in arginine transport in promastigotes, indicating that it is active and confirmed that LdAAP3 codes for an arginine transporter in parasite cells as well. LdAAP3 is novel as it shares a high level of homology with amino acid permeases from other trypanosomatidae but almost none with permeases from other phyla. The results of this work suggest that LdAAP3 might play a role in host-parasite interaction.

Coherent plant growth requires spatial integration of hormonal pathways and cell wall remodeling activities. However, the mechanisms governing sensitivity to hormones and how cell wall structure integrates with hormonal effects are poorly understood. We found that coordination between two types of epidermal root cells, hair and nonhair cells, establishes root sensitivity to the plant hormones brassinosteroids (BRs). While expression of the BR receptor BRASSINOSTEROID-INSENSITIVE1 (BRI1) in hair cells promotes cell elongation in all tissues, its high relative expression in nonhair cells is inhibitory. Elevated ethylene and deposition of crystalline cellulose underlie the inhibitory effect of BRI1. We propose that the relative spatial distribution of BRI1, and not its absolute level, fine-tunes growth.