The root system is fundamentally important for plant growth and survival because of its role in water and nutrient uptake. Therefore, plants rely on modulation of root system architecture (RSA) to respond to a changing soil environment. Although RSA is a highly plastic trait and varies both between and among species, the basic root system morphology and its plasticity are controlled by inherent genetic factors. These mediate the modification of RSA, mostly at the level of root branching, in response to a suite of biotic and abiotic factors. Recent progress in the understanding of the molecular basis of these responses suggests that they largely feed through hormone homeostasis and signaling pathways. Novel factors implicated in the regulation of RSA in response to the myriad endogenous and exogenous signals are also increasingly isolated through alternative approaches such as quantitative trait locus analysis.

Two classes of small RNAs, microRNAs and short-interfering RNA (siRNAs), have been extensively studied in plants and animals. In Arabidopsis, the capacity to uncover previously uncharacterized small RNAs by means of conventional strategies seems to be reaching its limits. To discover new plant small RNAs, we developed a protocol to mine an Arabidopsis nonannotated, noncoding EST database. Using this approach, we identified an endogenous small RNA, trans-acting short-interfering RNA-auxin response factor (tasiR-ARF), that shares a 21-and 22-nt region of sequence similarity with members of the ARF gene family. tasiR-ARF has characteristics of both short-interfering RNA and microRNA, recently defined as tasiRNA. Accumulation of trans-acting siRNA depends on DICER-LIKE1 and RNA-DEPENDENT RNA POLYMERASE6 but not RNA-DEPENDENT RNA POLYMERASE2. We demonstrate that tasiR-ARF targets three ARF genes, ARF2, ARF3͞ETT, and ARF4, and that both the tasiR-ARF precursor and its target genes are evolutionarily conserved. The identification of tasiRNA-ARF as a low-abundance, previously uncharacterized small RNA species proves our method to be a useful tool to uncover additional small regulatory RNAs.auxin response factor ͉ microRNA E ndogenous noncoding small RNAs 20-25 nt in length are important regulators of gene expression in both plants and animals (1-4). Recently, two major classes of small RNAs, microRNAs (miRNAs) and short-interfering RNAs (siRNAs), have been extensively studied. These two classes of small RNAs are generated from different types of precursor molecules, are processed through distinct biochemical pathways, and function in different biological contexts. miRNAs are processed from precursor molecules that are capable of forming double-stranded RNA (dsRNA) by intramolecular pairing (5) and act as regulatory factors during growth and development (1, 6, 7). In contrast, siRNAs are processed from either long bimolecular RNA duplexes produced by RNAdependent RNA polymerases (RDRs) or extended hairpins and function in several epigenetic and posttranscriptional silencing systems (8, 9). Two siRNA-generating pathways have been described: (i) an endogenous siRNA-generating pathway that requires RDR2 and triggers changes in the chromatin state of elements from which they derive (10) and (ii) an exogenous siRNA-generating pathway that requires RDR6 and targets viral RNAs (3). Despite the difference in the origin and generation of their precursors, both siRNAs and miRNAs require DICER proteins for processing, and both are assembled into the RNA-induced silencing complex (RISC) to target their complementary RNAs (3).Extensive efforts are being made to identify regulatory noncoding small RNA sequences, primarily focusing on miRNAs. Many miRNAs have been detected by using an established cloning strategy (11-14), although the low abundance and tissue-specific expression patterns of some make experimental identification difficult. Thus, different computational approaches have been developed to predict miRNA sequences based on t...

Brassinosteroid and auxin decisively influence plant development, and overlapping transcriptional responses to these phytohormones suggest an interaction between the two pathways. However, whether this reflects direct feedback or merely parallel inputs on common targets is unclear. Here we show that in Arabidopsis roots, this interaction is mediated by BREVIS RADIX (BRX), which is required for optimal root growth. We demonstrate that the brx phenotype results from a root-specific deficiency of brassinosteroid and is due to reduced, BRX-dependent expression of a rate-limiting enzyme in brassinosteroid biosynthesis. Unexpectedly, this deficiency affects the root expression level of approximately 15% of all Arabidopsis genes, but the transcriptome profile can be restored to wild type by brassinosteroid treatment. Thus, proper brassinosteroid levels are required for the correct expression of many more genes than previously suspected. Moreover, embryonic or post-embryonic brassinosteroid application fully or partially, respectively, rescues the brx phenotype. Further, auxin-responsive gene expression is globally impaired in brx, demonstrating that brassinosteroid levels are rate-limiting for auxin-responsive transcription. BRX expression is strongly induced by auxin and mildly repressed by brassinolide, which means that BRX acts at the nexus of a feedback loop that maintains threshold brassinosteroid levels to permit optimal auxin action.

Intercellular signaling is essential for the coordination of growth and development in higher plants. Although hundreds of putative receptors have been identified in Arabidopsis (Arabidopsis thaliana), only a few families of extracellular signaling molecules have been discovered, and their biological roles are largely unknown. To expand our insight into the developmental processes potentially regulated by ligand-mediated signal transduction pathways, we undertook a systematic expression analysis of the members of the Arabidopsis CLAVATA3/ESR-RELATED (CLE) small signaling polypeptide family. Using reporter constructs, we show that the CLE genes have distinct and specific patterns of promoter activity. We find that each Arabidopsis tissue expresses at least one CLE gene, indicating that CLE-mediated signaling pathways are likely to play roles in many biological processes during the plant life cycle. Some CLE genes that are closely related in sequence have dissimilar expression profiles, yet in many tissues multiple CLE genes have overlapping patterns of promoter-driven reporter activity. This observation, plus the general absence of detectable morphological phenotypes in cle null mutants, suggest that a high degree of functional redundancy exists among CLE gene family members. Our work establishes a community resource of CLE-related biological materials and provides a platform for understanding and ultimately manipulating many different plant signaling systems.

In Arabidopsis, interplay between nuclear auxin perception and trans-cellular polar auxin transport determines the transcriptional auxin response. In brevis radix (brx) mutants, this response is impaired, probably indirectly because of disturbed crosstalk between the auxin and brassinosteroid pathways. Here we provide evidence that BRX protein is plasma membrane-associated, but translocates to the nucleus upon auxin treatment to modulate cellular growth, possibly in conjunction with NGATHA class B3 domain-type transcription factors. Application of the polar auxin transport inhibitor naphthalene phthalamic acid (NPA) resulted in increased BRX abundance at the plasma membrane. Thus, nuclear translocation of BRX could depend on cellular auxin concentration or on auxin flux. Supporting this idea, NPA treatment of wild-type roots phenocopied the brx root meristem phenotype. Moreover, BRX is constitutively turned over by the proteasome pathway in the nucleus. However, a stabilized C-terminal BRX fragment significantly rescued the brx root growth phenotype and triggered a hypocotyl gain-of-function phenotype, similar to strong overexpressors of full length BRX. Therefore, although BRX activity is required in the nucleus, excess activity interferes with normal development. Finally, similar to the PIN-FORMED 1 (PIN1) auxin efflux carrier, BRX is polarly localized in vascular cells and subject to endocytic recycling. Expression of BRX under control of the PIN1 promoter fully rescued the brx short root phenotype, suggesting that the two genes act in the same tissues. Collectively, our results suggest that BRX might provide a contextual readout to synchronize cellular growth with the auxin concentration gradient across the root tip.

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BackgroundCellulose is an integral component of the plant cell wall and accounts for approximately forty percent of total plant biomass but understanding its mechanism of synthesis remains elusive. CELLULOSE SYNTHASE A (CESA) proteins function as catalytic subunits of a rosette-shaped complex that synthesizes cellulose at the plasma membrane. Arabidopsis thaliana and rice (Oryza sativa) secondary wall CESA loss-of-function mutants have weak stems and irregular or thin cell walls.ResultsHere, we identify candidates for secondary wall CESAs in Brachypodium distachyon as having similar amino acid sequence and expression to those characterized in A. thaliana, namely CESA4/7/8. To functionally characterize BdCESA4 and BdCESA7, we generated loss-of-function mutants using artificial microRNA constructs, specifically targeting each gene driven by a maize (Zea mays) ubiquitin promoter. Presence of the transgenes reduced BdCESA4 and BdCESA7 transcript abundance, as well as stem area, cell wall thickness of xylem and fibers, and the amount of crystalline cellulose in the cell wall.ConclusionThese results suggest BdCESA4 and BdCESA7 play a key role in B. distachyon secondary cell wall biosynthesis.

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