Brassinosteroid Regulates Cell Elongation by Modulating Gibberellin Metabolism in Rice

Brassinosteroid (BR) and gibberellin (GA) are two predominant hormones regulating plant cell elongation. A defect in either of these leads to reduced plant growth and dwarfism. However, their relationship remains unknown in rice (Oryza sativa). Here, we demonstrated that BR regulates cell elongation by modulating GA metabolism in rice. Under physiological conditions, BR promotes GA accumulation by regulating the expression of GA metabolic genes to stimulate cell elongation. BR greatly induces the expression of D18/GA3ox-2, one of the GA biosynthetic genes, leading to increased GA₁ levels, the bioactive GA in rice seedlings. Consequently, both d18 and loss-of-function GA-signaling mutants have decreased BR sensitivity. When excessive active BR is applied, the hormone mostly induces GA inactivation through upregulation of the GA inactivation gene GA2ox-3 and also represses BR biosynthesis, resulting in decreased hormone levels and growth inhibition. As a feedback mechanism, GA extensively inhibits BR biosynthesis and the BR response. GA treatment decreases the enlarged leaf angles in plants with enhanced BR biosynthesis or signaling. Our results revealed a previously unknown mechanism underlying BR and GA crosstalk depending on tissues and hormone levels, which greatly advances our understanding of hormone actions in crop plants and appears much different from that in Arabidopsis thaliana.     

RNA Interference Knockdown of BRASSINOSTEROID INSENSITIVE1 in Maize Reveals Novel Functions for Brassinosteroid Signaling in Controlling Plant Architecture

Brassinosteroids (BRs) are plant hormones involved in various growth and developmental processes. The BR signaling system is well established in Arabidopsis (Arabidopsis thaliana) and rice (Oryza sativa) but poorly understood in maize (Zea mays). BRASSINOSTEROID INSENSITIVE1 (BRI1) is a BR receptor, and database searches and additional genomic sequencing identified five maize homologs including duplicate copies of BRI1 itself. RNA interference (RNAi) using the extracellular coding region of a maize zmbri1 complementary DNA knocked down the expression of all five homologs. Decreased response to exogenously applied brassinolide and altered BR marker gene expression demonstrate that zmbri1-RNAi transgenic lines have compromised BR signaling. zmbri1-RNAi plants showed dwarf stature due to shortened internodes, with upper internodes most strongly affected. Leaves of zmbri1-RNAi plants are dark green, upright, and twisted, with decreased auricle formation. Kinematic analysis showed that decreased cell division and cell elongation both contributed to the shortened leaves. A BRASSINOSTEROID INSENSITIVE1-ETHYL METHANESULFONATE-SUPPRESSOR1-yellow fluorescent protein (BES1-YFP) transgenic line was developed that showed BR-inducible BES1-YFP accumulation in the nucleus, which was decreased in zmbri1-RNAi. Expression of the BES1-YFP reporter was strong in the auricle region of developing leaves, suggesting that localized BR signaling is involved in promoting auricle development, consistent with the zmbri1-RNAi phenotype. The blade-sheath boundary disruption, shorter ligule, and disrupted auricle morphology of RNAi lines resemble KNOTTED1-LIKE HOMEOBOX (KNOX) mutants, consistent with a mechanistic connection between KNOX genes and BR signaling.Brassinosteroids (BRs) are ubiquitous plant hormones that promote plant growth by regulating cell elongation and division (Clouse, 1996; Clouse et al., 1996). BRs have other diverse roles, including enhancing tracheary element differentiation, stimulating ATPase activity, controlling microtubule orientation, and controlling flowering time, fertility, and leaf development (Iwasaki and Shibaoka, 1991; Clouse et al., 1996; Li et al., 1996; Schumacher et al., 1999; Catterou et al., 2001; Oh et al., 2011). BRs also function in tolerance to both biotic and abiotic stresses such as extreme temperatures, drought, and pathogens (Krishna, 2003).Deficiencies in BR biosynthesis or signaling produce characteristic dwarf plant phenotypes (Clouse et al., 1996; Szekeres et al., 1996; Fujioka et al., 1997). Plant height is an important agricultural trait, as seen in the Green Revolution, where semidwarf mutants contributed to increased yields in small-grain crops (Salas Fernandez et al., 2009). BR-deficient dwarf rice (Oryza sativa) produced increased grain and biomass yields because the erect leaf habit allowed higher planting densities under field conditions (Sakamoto et al., 2006). In fact, Green Revolution Uzu barley (Hordeum vulgare) is based on a mutation of the UZU1 gene, which encodes a homolog of BRASSINOSTEROID INSENSITIVE1 (BRI1), a BR receptor (Chono et al., 2003).Genes functioning in BR pathways have been identified by the analysis of dwarf mutants in several species, including Arabidopsis (Arabidopsis thaliana) and rice. Arabidopsis bri1 mutants are shortened, have reduced apical dominance, and are male sterile (Clouse et al., 1996). BRI1 encodes a Leu-rich repeat (LRR) receptor-like kinase that is located in the plasma membrane and contains an extracellular domain responsible for BR binding, a transmembrane sequence, and a cytoplasmic protein kinase domain (Li and Chory, 1997; Vert et al., 2005; Belkhadir and Chory, 2006). The island domain and subsequent LRR 22 are critical for BR binding (Kinoshita et al., 2005; Hothorn et al., 2011; She et al., 2011). Phosphorylation of the conserved residues Ser-1044 and Thr-1049 in the kinase activation loop activates the BRI1 kinase (Wang et al., 2005), while dephosphorylation of BRI1 by PROTEIN PHOSPHATASE2A inhibits its function (Wu et al., 2011).BRI1 is partially redundant in BR signaling with related BRASSINOSTEROID INSENSITIVE1-LIKE RECEPTOR KINASE (BRL) paralogs, both in Arabidopsis and rice. In Arabidopsis, even though null alleles of brl1 or brl3 did not show obvious phenotypic defects in shoots, they enhanced the developmental defects of a weak bri1-5 mutant. In contrast to ubiquitously expressed BRI1, BRL1, BRL2, and BRL3 are tissue specific, mostly expressed in vascular tissues, while BRL1 and BRL3 are also expressed in root apices (Caño-Delgado et al., 2004; Zhou et al., 2004; Fàbregas et al., 2013). Both BRL1 and BRL3 can bind brassinolide (BL; Caño-Delgado et al., 2004). In rice, OsBRI1 is similar to the Arabidopsis BRI1 gene, and phenotypes of OsBRI1 rice mutants include dwarf plants with shortened internodes, erect leaves that are twisted and dark green, and photomorphogenesis in the dark (Yamamuro et al., 2000). There are three BR receptors in rice as well, and while OsBRI1 is universally expressed in all organs, OsBRL1 and OsBRL3 are expressed mostly in roots (Nakamura et al., 2006).To date, two mutant genes of the BR biosynthetic pathway have been reported in maize (Zea mays). A classic dwarf mutant, nana plant1 (na1), has a mutation in a DE-ETIOLATED2 homologous gene, which encodes a 5α-reductase enzyme in the BR biosynthesis pathway (Hartwig et al., 2011), while the brassinosteroid-dependent1 (brd1) gene encodes brassinosteroid C-6 oxidase (Makarevitch et al., 2012). The maize BR-deficient mutants have shortened internodes, twisted, dark green, erect leaves, and feminized male flowers (Hartwig et al., 2011; Makarevitch et al., 2012). However, no genes in BR signaling have yet been reported in maize. Understanding BR signaling in maize might help improve this important crop for the production of biofuels, biomass, and grain yield. Here, we took a transgenic RNA interference (RNAi) approach to generate maize plants partially deficient for BRI1. These knockdown lines demonstrate that BRI1 functions are generally conserved in maize compared with other plant species, but they also exhibit unique phenotypes, suggesting either that maize possesses novel BR-regulated developmental processes or that aspects of maize morphology reveal processes not evident in other plants.     

An Improved Simplified High-Sensitivity Quantification Method for Determining Brassinosteroids in Different Tissues of Rice and Arabidopsis

Quantification of brassinosteroids is essential and extremely important to study the molecular mechanisms of their physiological roles in plant growth and development. Herein, we present a simple, material and cost-saving high-performance method for determining endogenous brassinosteroids (BRs) in model plants. This new method enables simultaneous enrichment of a wide range of bioactive BRs such as brassinolide, castasterone, teasterone, and typhasterol with ion exchange solid-phase extraction and high-sensitivity quantitation of these BRs based on isotope dilution combined with internal standard approach. For routine analysis, the consumption of plant materials was reduced to one-twentieth of previously reported and the overall process could be completed within 1 day compared with previous 3 to 4 days. The strategy was validated by profiling BRs in different ecotypes and mutants of rice (Oryza sativa) and Arabidopsis (Arabidopsis thaliana), and the BR distributions in different model plants tissues were determined with the new method. The method allows plant physiologists to monitor the dynamics and distributions of BRs with 1 gram fresh weight of model plant tissues, which will speed up the process for the molecular mechanism research of BRs with these model plants in future work.Brassinosteroids (BRs) have been considered as the sixth class of endogenous plant hormones with wide occurrence across the plant kingdom (Bajguz and Tretyn, 2003). BRs play a key role in a variety of physiological processes, such as cell elongation, vascular differentiation, reproductive development, photomorphogenesis, stress tolerance, and so on (Hayat, 2010). Recently, it was found that BR deficiency could increase grain yield in rice (Oryza sativa) by more than 30%, which showed a food security-enhancing potential and guided new green revolution in the future (Sakamoto et al., 2006; Wu et al., 2008). Since BRs were first isolated and identified from rape (Brassica napus) pollen in 1970s (Mitchell et al., 1970; Grove et al., 1979), the natural occurrence of more than 60 BRs in a large quantity of plant species has been reported (Hayat, 2010). To date, research on the occurrence of BRs in different plants, physiological properties, and their action modes has made much progress (Fujioka and Yokota, 2003; Symons et al., 2008; Kim and Wang, 2010; Tang et al., 2010; Tong and Chu, 2012). However, so far, only limited information was obtained to understand the molecular mechanism of the physiological role of BRs. For example, although the biosynthetic pathway of C₂₈ BRs has been well established, the biosynthesis of C₂₇ and C₂₉ BRs remains unclear, and some intermediates on their biosynthetic pathways still need to be elucidated (Noguchi et al., 2000; Fujita et al., 2006). The plant physiology research of BRs is speeded up by employing BR mutants on biosynthesis and signaling pathways (Yamamuro et al., 2000; Hong et al., 2003; Kwon and Choe, 2005; Tanabe et al., 2005); however, a simple, high-sensitivity screening, detection, and quantification method for BR analysis is still a bottleneck technique for in-depth studying of the role of BRs during the life cycle of plants.In the past 20 years, most of the detection and identification processes of BRs could be described briefly as the following steps. The harvested plant materials were lyophilized and then ground to a fine powder, followed by the CH₃OH/CHCl₃ extraction. The concentrate was then partitioned with the CHCl₃/H₂O system three times. After that, the CHCl₃ fraction was subjected to a silica gel column for BR enrichment, and the collected BRs-containing fraction was purified with Sephadex LH-20 column and Sep-Pak Plus C18 cartridge in sequence. At last, the collected fractions were purified with preparative HPLC and then derivatized for analysis with gas chromatography-mass spectrometry (MS) under selected ion monitoring mode (Hong et al., 2005; Nomura et al., 2005; Kim et al., 2006). So far, this protocol has been proven to be workable in most cases and provided a great quantity of valuable data for plant physiological research (Hwang et al., 2006; Lee et al., 2010). However, at least more than 20 g of plant materials were consumed for quantifying/identifying BRs in one plant sample without replicates (Hong et al., 2005; Bancos et al., 2006; Kim et al., 2006), and it is difficult to collect sufficient plant tissues for BR measurement in some rare model plant mutants. In addition, the method involved multiple tedious and labor-intensive steps, which might result in poor recovery and low sensitivity, especially for some labile BR intermediates. The traditional method took one person about 3 to 4 d to treat one batch of samples. Most of the BR measurement experiments were performed without biological replicates using traditional methods due to the disadvantages mentioned above, which discounted the reliability of the results. Therefore, a simple, rapid, and sensitive analysis method for BRs is in urgent need, along with the development of BR research.Recently, several efforts were made to improve the BR determination (Svatos et al., 2004; Huo et al., 2012). The consumption of plant material was reduced to 2 g after modifying the BRs with a new derivatization reagent for further ultra-performance liquid chromatography (UPLC)-multiple reaction monitoring (MRM)-MS detection. However, the purification process consisting of deproteinization and multiple solid-phase extraction (SPE) steps was still quite tedious and couldn’t guarantee covering the four most important bioactive BRs, including brassinolide (BL), castasterone (CS), teasterone (TE), and typhasterol (TY; Fig. 1). In our previous study (Xin et al., 2013), we reported a simple, convenient, and high-sensitivity method for detection of endogenous BRs from real plant materials based on the dual role of specific boronate affinity. Although it was the first time to measure multiple BRs in subgram plant materials and the time duration of the method decreased to one-third of that previously reported, the synthesis of boronate affinity-functionalized magnetic nanoparticles made the method difficult to follow in biological laboratories for routine analysis.Open in a separate windowFigure 1.Chemical structure of four major bioactive BRs.BRs are neutral steroid compounds with a common four-ring cholestane skeleton and hydroxyl groups on A ring and/or the side chain linked to D ring. Especially, the vicinal diol moieties on C₂₂ and C₂₃ sites of BL, CS, TY, and TE allow these bioactive BRs to be derivatized with ionizable reagents for MS response enhancement. Considering the unique physicochemical properties of BRs, we herein developed a simplified high-sensitivity analytical method based on mixed-mode anion exchange (MAX)-cation exchange (MCX) solid phase extraction (SPE) purification, vicinal diol derivatization combined with UPLC-MRM3-MS detection for quantification of BL, CS, TE, and TY in model plants (Fig. 2). The performance of the method was demonstrated by determination of BRs in different tissues of both wild-type and mutant Arabidopsis (Arabidopsis thaliana) and rice.Open in a separate windowFigure 2.Simplified high-sensitivity protocol for quantitative analysis of BRs. IS, Internal standards. [See online article for color version of this figure.]     

Cytochrome P450 CYP89A9 Is Involved in the Formation of Major Chlorophyll Catabolites during Leaf Senescence in Arabidopsis

Nonfluorescent chlorophyll catabolites (NCCs) were described as products of chlorophyll breakdown in Arabidopsis thaliana. NCCs are formyloxobilin-type catabolites derived from chlorophyll by oxygenolytic opening of the chlorin macrocycle. These linear tetrapyrroles are generated from their fluorescent chlorophyll catabolite (FCC) precursors by a nonenzymatic isomerization inside the vacuole of senescing cells. Here, we identified a group of distinct dioxobilin-type chlorophyll catabolites (DCCs) as the major breakdown products in wild-type Arabidopsis, representing more than 90% of the chlorophyll of green leaves. The molecular constitution of the most abundant nonfluorescent DCC (NDCC), At-NDCC-1, was determined. We further identified cytochrome P450 monooxygenase CYP89A9 as being responsible for NDCC accumulation in wild-type Arabidopsis; cyp89a9 mutants that are deficient in CYP89A9 function were devoid of NDCCs but accumulated proportionally higher amounts of NCCs. CYP89A9 localized outside the chloroplasts, implying that FCCs occurring in the cytosol might be its natural substrate. Using recombinant CYP89A9, we confirm FCC specificity and show that fluorescent DCCs are the products of the CYP89A9 reaction. Fluorescent DCCs, formed by this enzyme, isomerize to the respective NDCCs in weakly acidic medium, as found in vacuoles. We conclude that CYP89A9 is involved in the formation of dioxobilin-type catabolites of chlorophyll in Arabidopsis.     

Comprehensive Protein-Based Artificial MicroRNA Screens for Effective Gene Silencing in Plants

Artificial microRNA (amiRNA) approaches offer a powerful strategy for targeted gene manipulation in any plant species. However, the current unpredictability of amiRNA efficacy has limited broad application of this promising technology. To address this, we developed epitope-tagged protein-based amiRNA (ETPamir) screens, in which target mRNAs encoding epitope-tagged proteins were constitutively or inducibly coexpressed in protoplasts with amiRNA candidates targeting single or multiple genes. This design allowed parallel quantification of target proteins and mRNAs to define amiRNA efficacy and mechanism of action, circumventing unpredictable amiRNA expression/processing and antibody unavailability. Systematic evaluation of 63 amiRNAs in 79 ETPamir screens for 16 target genes revealed a simple, effective solution for selecting optimal amiRNAs from hundreds of computational predictions, reaching ∼100% gene silencing in plant cells and null phenotypes in transgenic plants. Optimal amiRNAs predominantly mediated highly specific translational repression at 5′ coding regions with limited mRNA decay or cleavage. Our screens were easily applied to diverse plant species, including Arabidopsis thaliana, tobacco (Nicotiana benthamiana), tomato (Solanum lycopersicum), sunflower (Helianthus annuus), Catharanthus roseus, maize (Zea mays) and rice (Oryza sativa), and effectively validated predicted natural miRNA targets. These screens could improve plant research and crop engineering by making amiRNA a more predictable and manageable genetic and functional genomic technology.