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- Materials and Methods
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Cyclophilins (CyPs) constitute a large family of proteins that are present in a broad range of organisms, from bacteria to humans (Galat, 1999). The first CyP, CyP A, was identified as the specific target for the immunosuppressant cyclosporin A in mammalian T-cells (Handschumacher et al., 1984). CyPs possess peptidyl-prolyl cis-trans isomerase (rotamase) activity that catalyzes the rotation of X-proline peptide bonds from a cis to a trans conformation, a rate-limiting step in the protein-folding process (Galat, 1993). As potential molecular chaperones, CyPs play important roles in a variety of cellular processes, including protein trafficking (Freskgard et al., 1992), cell division (Faure et al., 1998), transcriptional regulation (Rycyzyn & Clevenger, 2002), signal transduction (Brazin et al., 2002), and RNA processing and spliceosome assembly (Gullerova et al., 2006). In higher plants, CyPs were first identified in tomato, maize, and oilseed rape (Gasser et al., 1990). Since then, CyP genes have been discovered in many other plant species, such as rice (Buchholz et al., 1994; Kumari et al., 2009), bean (Kinoshita & Shimazaki, 1999), and wheat (Li et al., 2010). In Arabidopsis, 29 CyP genes have been identified (Romano et al., 2004). Plant CyPs exist in all subcellular compartments and participate in protection from photo-damage (Dominguez-Solis et al., 2008), assembly of photosynthesis system II (Fu et al., 2007), and plant responses to biotic (Coaker et al., 2005) and abiotic stresses (Luan et al., 1994; Chen et al., 2007). Several CyPs have been found to be involved in the control of plant growth and development. A loss-of-function mutation in the CyP40 gene causes the precocious expression of the adult vegetative trait, but does not affect flowering time in Arabidopsis (Berardini et al., 2001). Further study indicated that cyp40 mutation caused the elevated expression of miRNA165-regulated members of the SPL family of transcription factors and thereby accelerated the phase transition from juvenile to adult (Smith et al., 2009). Li et al. (2007) reported that a WD40 domain-containing CyP, CyP71, regulates gene repression and organogenesis in Arabidopsis. The cyp71 null mutant exhibits reduced apical meristem activity, delayed and abnormal lateral organ formation, and arrested root growth. CyP71 protein is localized in the nucleus and affects the amount of histone methylation at the loci of homeotic genes KNAT1 and STM, which are two key regulators of shoot meristem activity. gaid is a wheat mutant with dwarf phenotype (Li et al., 2010). This mutant overaccumulates a CyP-like protein, TaCYP20-2, and overexpression of TaCYP20-2 in wildtype (WT) wheat resulted in a similar dwarf phenotype as gaid. The tomato auxin-resistant mutant diageotropica (dgt), which carries a mutation in the LeCYP1 gene, displays a pleiotropic phenotype including a slow gravitropic response, lack of lateral roots, and reduction of apical dominance (Oh et al., 2006).
Plant architecture is an important agronomic trait and is the most useful characteristic for identifying plant species (Wang & Li, 2007). It is defined by plant height and by the numbers and spatial arrangement of shoot branches and inflorescences. Alteration of plant architecture, such as plant height and tillering number in rice, can have a dramatic effect on crop yield. Since the 1960s, introduction of dwarf wheat and rice cultivars has greatly improved plant resistance to wind and rain and has increased grain yields (Hedden, 2003). Analyses of these dwarf cultivars and some dwarf mutants of Arabidopsis, pea, and tomato have led to the discoveries of several important genes involved in the control of plant height and shoot branching. It has become clear that the plant hormone gibberellic acid (GA) and brassinosteroid are two major regulators of plant stem elongation. Mutants defective in either the biosynthetic or signaling pathways of these two hormones exhibit a dwarf phenotype (Wang & Li, 2007). In Arabidopsis and rice, GA is perceived by GA receptors (Sun, 2010; Ueguchi-Tanaka & Matsuoka, 2010). Upon binding of GA, the GA-bound receptors interact with a DELLA protein in rice (Murase et al., 2008) or five DELLA proteins in Arabidopsis (Jiang & Fu, 2007; Harberd et al., 2009; Ariizumi et al., 2010). These DELLA proteins serve as negative regulators of GA responses. In the absence of GA, they suppress downstream responses by interacting with multiple regulatory proteins such as basic helix–loop–helix (bHLH) transcription factors PHYTOCHROME INTERACTING FACTORS (Harberd et al., 2009), ALCATRAZ (Arnaud et al., 2010), and GRAS protein SCARECROW-LOKE 3 (Heo et al., 2011). The interaction between the GA-bound receptors and the DELLA proteins causes DELLA proteins to be ubiquitinated via the GID2-based SCF complex in rice (Gomi et al., 2004) or via the SLY1-based SCF complex in Arabidopsis (McGinnis et al., 2003). The ubiquitinated DELLA proteins will then be targeted to 26S proteasome for degradation. The GA-mediated degradation of DELLA proteins thus releases GA responses, which include promotion of seed germination, stem elongation, flowering, and flower and silique development. In Arabidopsis, a gain-of-function mutation in one of the DELLA proteins, GAI, renders it resistant to GA-mediated degradation and also results in a dwarf phenotype (Peng et al., 1997). The orthologs of Arabidopsis GAI in wheat (Rht) and maize (D8) were found to be the ‘Green revolution’ genes that greatly enhanced grain yield in the 1960s (Peng et al., 1999). The GA-mediated degradation of Rht protein is also suppressed in the wheat dwarf mutant gaid (Li et al., 2010). By contrast, the functional disruption of the SLR1 gene that encodes the only DELLA protein in rice causes the plants to display a constitutive GA response, resulting in a slender phenotype (Ikeda et al., 2001).
We report here that a gain-of-function mutation in a highly conserved Arabidopsis CyP gene, ROC1, causes a dramatic alteration of plant architecture. The roc1 mutation does not seem to interfere with GA biosynthesis or signaling; however, GA signaling antagonizes the effect of roc1 mutation on stem elongation. Our work provides a useful genetic material that may help with the further identification of molecular components involved in the control of plant architecture.
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- Materials and Methods
- Supporting Information
In this work, we identified an Arabidopsis mutant, roc1, with altered plant architecture. The heterozygote mutant plants (roc1/+) are shorter and produce more shoot branches than the WT. The homozygote mutant plants (roc1/roc1) cannot bolt. In roc1/+ plants, stem epidermal cells are 50% shorter than in the WT, providing a cellular basis for reduced stem elongation. The roc1 mutant phenotypes are strongly affected by temperature and photoperiod. The flowering time and development of flower organs and siliques, however, are not altered in roc1/+ or roc1/roc1 plants, suggesting that the mutation does not affect reproductive development. Our genetic and molecular analyses indicate that the mutant phenotypes were caused by a gain-of-function mutation in a cyclophilin gene ROC1. ROC1 was cloned and characterized as a cytosolic CyP in Arabidopsis in 1994 (Lippuner et al., 1994). So far, the only known biological function of ROC1 is its role in a plant–pathogen interaction (Coaker et al., 2005). Specifically, when the bacterium Pseudomonas syringae infects Arabidopsis plants, it delivers an effector protein AvrRpts (a cysteine protease) into host cells. After it is delivered into host cells, AvrRpts is activated by ROC1, which binds to AvrRpts at four sites and properly folds it by peptidyl-prolyl cis/trans isomerization (Coaker et al., 2006; Aumüller et al., 2010). Activation of AvrRpts leads to the cleavage of the Arabidopsis protein RIN4, which triggers the activation of disease-resistance gene RPS2-mediated defense response. It has long been thought that plant growth and defense response are intimately connected and that there are some tradeoffs between these two processes (Heil & Baldwin, 2002). Retarded growth is sometimes exhibited by mutants with constitutive defense responses, possibly because of resource reallocation from growth to defense or the metabolic burden of defense responses on plants, or both. For example, plants with a loss-of-function mutation in the BON1 gene, which encodes a copine-like protein, exhibit precocious cell death and enhanced disease resistance at low temperatures (Jambunathan et al., 2001). The bon1 plants also have a severe dwarf phenotype (Hua et al., 2001). The disruption of the BON1 function activates a resistance (R) gene, SNC1, leading to constitutive defense responses and retarded growth (Yang & Hua, 2004). These are also the phenotypes of snc1 mutant plants that carry a gain-of-function mutation in the SNC1 gene, resulting in the activation of SNC1 (Zhang et al., 2003). Similarly, the Arabidopsis mutant cpr30 displays both dwarfism and constitutively activated defense responses (Guo et al., 2009). Our mapping of the second locus that is linked to roc1 mutant phenotypes narrowed the candidate gene to a 140 kb region on chromosome 1. In this region, we found five putative R genes. We speculate that the point mutation in the ROC1 protein may alter its enzymatic activity, resulting in the activation of one of these five R genes, thus causing plants to display a constitutive defense response. This R gene may be specific to the Col ecotype. Thus, when a roc1 plant (in the Col ecotype) was crossed to a plant of the Ler ecotype, the ortholog of this R gene in the Ler plant functions as a suppressor of the roc1 mutant phenotypes. The same scenario was previously found for SNC1-mediated plant growth and disease resistance. SNC1 is specific to the Col ecotype, and the same SNC1 sequence is absent in the Wassilewskija (Ws) ecotype (Yang & Hua, 2004). As a consequence, Ws plants with a loss-of-function mutation in the BON1 gene were unable to display the mutant phenotypes as observed for the Col plants. Previous reports have indicated that high temperature can suppress many R gene-mediated dwarf phenotypes, including those observed for bon1, snc1, and cpr30 (Zhang et al., 2003; Yang & Hua, 2004; Guo et al., 2009), and low light reduced cell death associated with disease resistance (Dietrich et al., 1994; Xiao et al., 2003). roc1 mutant phenotypes were also inhibited by high temperature and low light, providing more evidence for the hypothesis that the growth defects of the roc1 mutant result from activated defense responses. To further support this hypothesis, we must analyze the defense-related responses of roc1 plants and determine which of the five putative R genes is the target of the roc1 protein.
Alternatively, WT ROC1 protein may not play any roles in regulating plant growth and development. Instead, the mutation in the ROC1 gene may cause the roc1 protein to have a novel function in which it interacts with new targets that affect stem elongation. These new targets could include the molecular components involved in GA biosynthesis or signaling, or the proteins directly involved in the control of cell expansion. This hypothesis is supported by the fact that both ROC1-overexpressing or RNAi lines do not display altered growth phenotypes. Thus, it will interesting to determine how this mutation affects the ROC1 protein structure and its target specificity. In addition, our results indicate that the gene mutation enables roc1 proteins to respond to GA signal in terms of their protein stability, establishing a possible link between the new function of roc1 protein and GA signaling.
The plant hormone GA plays a crucial role in regulating stem elongation. Our work shows that application of exogenous GA3 cannot rescue the roc1 mutant phenotypes, indicating that the roc1 mutant phenotypes do not result from a deficiency in GA biosynthesis. In addition, the seed germination rates of roc1/+ or roc1/roc1 plants were similar to that of the WT (data not shown), further indicating that the mutant plants were not deficient in GA. An alternative explanation, that the roc1 mutation, like the overexpression of TaCYP20-2 in wheat (Li et al., 2010), makes plants insensitive to GA signaling also seems incorrect for the following reasons. First, GA3 treatment led to further suppression of stem elongation in roc1/+ plants, indicating that the mutant plants were able to sense the GA signal, even though the GA effect on growth was contrary to what was expected. Secondly, the leaf morphologies of GA3-treated roc1/+ and roc1/roc1 plants resembled that of GA3-treated WT plants, suggesting that responsiveness of leaves to GA signal in the mutant plants was not impaired. Thirdly, flowering times of the WT and the mutant plants were similar. Finally, the GA-mediated degradation of a key GA signaling component, RGA, is not affected in roc1 plants. All these results indicate that the impairment of stem elongation in roc1/+ and roc1/roc1 plants is not a result of plant insensitivity to GA signals.
To explain the inhibitory effect of GA3 on stem elongation in roc1/+ plants, we hypothesize the following model. Under normal growth conditions, the WT ROC1 proteins do not function in the control of stem elongation. The point mutation in the ROC1 gene, however, changes the conformation of its encoded protein and causes the protein (roc1) to have a new function. In this new function, roc1 protein alters the activity of new target proteins that are directly involved in the control of stem elongation (Fig. 7). The suppressive effect of roc1 protein on stem elongation is proportional to the quantity of roc1 protein expressed – that is, higher expression leads to more severe mutant phenotypes. The effects of the roc1 protein on its downstream targets, however, are antagonized by the DELLA proteins. We propose that treatment of the mutant plants with GA3 caused the degradation of DELLA proteins, which would tend to enhance stem elongation. Treatment of the mutant plants with GA3, however, also increased the abundance of roc1 proteins (perhaps as a result of the elimination of the antagonistic effect of DELLA proteins and some other unknown mechanisms), which would inhibit stem elongation. As a consequence, the phenotype of GA3-treated roc1 plants will depend on these two opposite effects on stem elongation. If the effect of enhanced roc1 accumulation is greater than that of the degradation of DELLA proteins, the plants will exhibit a further suppression of stem elongation. In the case of the roc1/gai double mutant, however, the mutated GAI protein might permanently block the activity of roc1 protein (through either direct or indirect interactions). Thus, the roc1/gai plants display only the phenotypes of gai.
Figure 7. A model for the interactions between the roc1 mutation and gibberellic acid (GA) signaling in the control of stem elongation in Arabidopsis thaliana.
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Finally, we want to point out that, although the WT ROC1 protein may not play a direct role in regulating plant growth and development, the gain-of-function roc1 mutant will be useful for further identification of novel molecular components involved in the control of plant architecture. This may be achieved by final identification of the genetic suppressor of roc1 described in this study. Our molecular mapping for the second genetic locus that is linked to the roc1 phenotype implied that this suppressor is probably an ecotype-specific R gene. If this inference can be confirmed, it will help to explain how the activation of an R gene affects plant development. Alternatively, molecular and biochemical approaches, such as yeast two-hybrid assay and purification of the roc1-containing protein complex, could be used to identify the roc1 interacting proteins and thereby to gain new insights into the molecular basis of plant architecture. In addition, the introduction of mutated ROC1 proteins or the mutated form of their orthologs into crops may create new and useful cultivars for breeding.