Genome-Wide Control of Polyadenylation Site Choice by CPSF30 in Arabidopsis

The Arabidopsis thaliana ortholog of the 30-kD subunit of the mammalian Cleavage and Polyadenylation Specificity Factor (CPSF30) has been implicated in the responses of plants to oxidative stress, suggesting a role for alternative polyadenylation. To better understand this, poly(A) site choice was studied in a mutant (oxt6) deficient in CPSF30 expression using a genome-scale approach. The results indicate that poly(A) site choice in a large majority of Arabidopsis genes is altered in the oxt6 mutant. A number of poly(A) sites were identified that are seen only in the wild type or oxt6 mutant. Interestingly, putative polyadenylation signals associated with sites that are seen only in the oxt6 mutant are decidedly different from the canonical plant polyadenylation signal, lacking the characteristic A-rich near-upstream element (where AAUAAA can be found); this suggests that CPSF30 functions in the handling of the near-upstream element. The sets of genes that possess sites seen only in the wild type or mutant were enriched for those involved in stress and defense responses, a result consistent with the properties of the oxt6 mutant. Taken together, these studies provide new insights into the mechanisms and consequences of CPSF30-mediated alternative polyadenylation.     

Calmodulin interacts with and regulates the RNA-binding activity of an Arabidopsis polyadenylation factor subunit

The Arabidopsis (Arabidopsis thaliana) gene that encodes the probable ortholog of the 30-kD subunit of the mammalian cleavage and polyadenylation specificity factor (CPSF) is a complex one, encoding small (approximately 28 kD) and large (approximately 68 kD) polypeptides. The small polypeptide (AtCPSF30) corresponds to CPSF30 and is the focus of this study. Recombinant AtCPSF30 was purified from Escherichia coli and found to possess RNA-binding activity. Mutational analysis indicated that an evolutionarily conserved central core of AtCPSF30 is involved in RNA binding, but that RNA binding also requires a short sequence adjacent to the N terminus of the central core. AtCPSF30 was found to bind calmodulin, and calmodulin inhibited the RNA-binding activity of the protein in a calcium-dependent manner. Mutational analysis showed that a small part of the protein, again adjacent to the N terminus of the conserved core, is responsible for calmodulin binding; point mutations in this region abolished both binding to and inhibition of RNA binding by calmodulin. Interestingly, AtCPSF30 was capable of self-interactions. This property also mapped to the central conserved core of the protein. However, calmodulin had no discernible effect on the self-association. These results show that the central portion of AtCPSF30 is involved in a number of important functions, and they raise interesting possibilities for both the interplay between splicing and polyadenylation and the regulation of these processes by stimuli that act through calmodulin.     

The Arabidopsis ortholog of the 77 kDa subunit of the cleavage stimulatory factor (AtCstF-77) involved in mRNA polyadenylation is an RNA-binding protein

The 77 kDa subunit of the polyadenylation cleavage stimulation factor (CstF77) is important in messenger RNA 3′ end processing. Previously, we demonstrated that AtCstF77 interacts with AtCPSF30, the Arabidopsis ortholog of the 30 kDa subunit of the Cleavage and Polyadenylation Specificity Factor. In further dissecting this interaction, it was found that the C-terminus of AtCstF77 interacts with AtCPSF30. Remarkably, we also found that the C-terminal domain of AtCstF77 possesses RNA-binding ability. These studies therefore reveal AtCstF77 to be an RNA-binding protein, adding yet another RNA-binding activity to the plant polyadenylation complex. This raises interesting questions as to the means by which RNAs are recognized during mRNA 3′ end formation in plants.

Structured summary:

MINT-7712550: AtCstF77 (uniprotkb:Q8LKG5) binds (MI:0407) to AtCPSF30 (uniprotkb:A9LNK9) by pull down (MI:0096)     

A novel endonuclease activity associated with the Arabidopsis ortholog of the 30-kDa subunit of cleavage and polyadenylation specificity factor

The polyadenylation of messenger RNAs is mediated by a multi-subunit complex that is conserved in eukaryotes. Among the most interesting of these proteins is the 30-kDa-subunit of the Cleavage and Polyadenylation Specificity Factor, or CPSF30. In this study, the Arabidopsis CPSF30 ortholog, AtCPSF30, is characterized. This protein possesses an unexpected endonucleolytic activity that is apparent as an ability to nick and degrade linear as well as circular single-stranded RNA. Endonucleolytic action by AtCPSF30 leaves RNA 3′ ends with hydroxyl groups, as they can be labeled by RNA ligase with [³²P]-cytidine-3′,5′-bisphosphate. Mutations in the first of the three CCCH zinc finger motifs of the protein abolish RNA binding by AtCPSF30 but have no discernible effects on nuclease activity. In contrast, mutations in the third zinc finger motif eliminate the nuclease activity of the protein, and have a modest effect on RNA binding. The N-terminal domain of another Arabidopsis polyadenylation factor subunit, AtFip1(V), dramatically inhibits the nuclease activity of AtCPSF30 but has a slight negative effect on the RNA-binding activity of the protein. These results indicate that AtCPSF30 is a probable processing endonuclease, and that its action is coordinated through its interaction with Fip1.     

Analysis the role of arabidopsis <Emphasis Type="Italic">CKRC6/ASA1</Emphasis> in auxin and cytokinin biosynthesis

The crosstalk between auxin and cytokinin (CK) is important for plant growth and development, although the underlying molecular mechanisms remain unclear. Here, we describe the isolation and characterization of a mutant of Arabidopsis Cytokinin-induced Root Curling 6 (CKRC6), an allele of ANTHRANILATE SYNTHASE ALPHA SUBUNIT 1 (ASA1) that encodes the á-subunit of AS in tryptophan (Trp) biosynthesis. The ckrc6 mutant exhibits root gravitropic defects and insensitivity to both CK and the ethylene precursor 1-aminocyclopropane-1-carboxylicacid (ACC) in primary root growth. These defects can be rescued by exogenous indole-3-acetic acid (IAA) or tryptophan (Trp) supplementation. Furthermore, our results suggest that the ckrc6 mutant has decreased IAA content, differential expression patterns of auxin biosynthesis genes and CK biosynthesis isopentenyl transferase (IPT) genes in comparison to wild type. Collectively, our study shows that auxin controls CK biosynthesis based on that CK sensitivity is altered in most auxin-resistant mutants and that CKs promote auxin biosynthesis but inhibit auxin transport and response. Our results also suggest that CKRC6/ASA1 may be located at an intersection of auxin, CK and ethylene metabolism and/or signaling.     

Compensation for a Mutated Auxin Biosynthesis Gene of Agrobacterium Ti Plasmid A66 in Nicotiana glutinosa Does Not Result from Increased Auxin Accumulation

Nicotiana glutinosa compensated for a mutated tumor-morphology-shooty (tms) (auxin biosynthesis) locus of Agrobacterlum tumefaciens strain A66 and showed the same virulent tumor response to infection by strain A66 or the wild-type strain A6. Cloned cell lines transformed by strains A6 or A66 were fully hormone independent in culture and grew rapidly as friable, unorganized tissues on hormone-free growth medium. Growth of N. glutinosa tumor cells was inhibited by addition of α-naphthaleneacetic acid to the growth medium, and A6- and A66-transformed cells showed similar dose responses to this auxin. On the other hand, A6-transformed cells contained much higher levels of indole-3-acetic acid (IAA) and 1-aminocyclopropane-1-carboxylic acid (ACC) than A66-transformed cells. Differences in IAA and ACC levels in N. glutinosa tumor lines were consistent with the expected activity of the tms locus and were quantitatively similar to results obtained previously with A6- and A66-transformed cells of Nicotiana tabacum, which does not compensate for mutated tms genes. Thus, compensation for mutated tms genes in N. glutinosa did not result from increased auxin accumulation and did not appear to be related to the capacity of this host for auxin biosynthesis.     

The Diageotropica Gene Differentially Affects Auxin and Cytokinin Responses throughout Development in Tomato

The interactions between the plant hormones auxin and cytokinin throughout plant development are complex, and genetic investigations of the interdependency of auxin and cytokinin signaling have been limited. We have characterized the cytokinin sensitivity of the auxin-resistant diageotropica (dgt) mutant of tomato (Lycopersicon esculentum Mill.) in a range of auxin- and cytokinin-regulated responses. Intact, etiolated dgt seedlings showed cross-resistance to cytokinin with respect to root elongation, but cytokinin effects on hypocotyl growth and ethylene synthesis in these seedlings were not impaired by the dgt mutation. Seven-week-old, green wild-type and dgt plants were also equally sensitive to cytokinin with respect to shoot growth and hypocotyl and internode elongation. The effects of cytokinin and the dgt mutation on these processes appeared additive. In tissue culture organ regeneration from dgt hypocotyl explants showed reduced sensitivity to auxin but normal sensitivity to cytokinin, and the effects of cytokinin and the mutation were again additive. However, although callus induction from dgt hypocotyl explants required auxin and cytokinin, dgt calli did not show the typical concentration-dependent stimulation of growth by either auxin or cytokinin observed in wild-type calli. Cross-resistance of the dgt mutant to cytokinin thus was found to be limited to a small subset of auxin- and cytokinin-regulated growth processes affected by the dgt mutation, indicating that auxin and cytokinin regulate plant growth through both shared and separate signaling pathways.     

Pleiotropic and nonredundant effects of an auxin importer in Setaria and maize

Directional transport of auxin is critical for inflorescence and floral development in flowering plants, but the role of auxin influx carriers (AUX1 proteins) has been largely overlooked. Taking advantage of available AUX1 mutants in green millet (Setaria viridis) and maize (Zea mays), we uncover previously unreported aspects of plant development that are affected by auxin influx, including higher order branches in the inflorescence, stigma branch number, glume (floral bract) development, and plant fertility. However, disruption of auxin flux does not affect all parts of the plant, with little obvious effect on inflorescence meristem size, time to flowering, and anther morphology. In double mutant studies in maize, disruptions of ZmAUX1 also affect vegetative development. A green fluorescent protein (GFP)-tagged construct of the Setaria AUX1 protein Sparse Panicle1 (SPP1) under its native promoter showed that SPP1 localizes to the plasma membrane of outer tissue layers in both roots and inflorescences, and accumulates specifically in inflorescence branch meristems, consistent with the mutant phenotype and expected auxin maxima. RNA-seq analysis indicated that most gene expression modules are conserved between mutant and wild-type plants, with only a few hundred genes differentially expressed in spp1 inflorescences. Using clustered regularly interspaced short palindromic repeats (CRISPR)–Cas9 technology, we disrupted SPP1 and the other four AUX1 homologs in S. viridis. SPP1 has a larger effect on inflorescence development than the others, although all contribute to plant height, tiller formation, and leaf and root development. The AUX1 importers are thus not fully redundant in S. viridis. Our detailed phenotypic characterization plus a stable GFP-tagged line offer tools for future dissection of the function of auxin influx proteins.

Mutations in a single auxin importer gene uncover broad and unexpected effects in nearly all aspects of the development of shoots, inflorescences, and flowers.     

The 73 kD Subunit of the cleavage and polyadenylation specificity factor (CPSF) complex affects reproductive development in Arabidopsis

The cleavage and polyadenylation specificity factor (CPSF) is an important multi-subunit component of the mRNA 3′-end processing apparatus in eukaryotes. The Arabidopsis genome contains five genes encoding CPSF homologues (AtCPSF160, AtCPSF100, AtCPSF73-I, AtCPSF73-II and AtCPSF30). These CPSF homologues interact with each other in a way that is analogous to the mammalian CPSF complex or their yeast counterparts, and also interact with the Arabidopsis poly(A) polymerase (PAP). There are two CPSF73 like proteins (AtCPSF73-I and AtCPSF73-II) that share homology with the 73 kD subunit of the mammalian CPSF complex. AtCPSF73-I appears to correspond to the functionally characterized mammalian CPSF73 and its yeast counterpart. AtCPSF73-II was identified as a novel protein with uncharacterized protein homologues in other multicellular organisms, but not in yeast. Both of the AtCPSF73 proteins are targeted in the nucleus and were found to interact with AtCPSF100. They are also essential since knockout or knockdown mutants are lethal. In addition, the expression level of AtCPSF73-I is critical for Arabidopsis development because overexpression of AtCPSF73-I is lethal. Interestingly, transgenic plants carrying an additional copy of the AtCPSF73-I gene, that is, the full-length cDNA under the control of its native promoter, appeared normal but were male sterile due to delayed anther dehiscence. In contrast, we previously demonstrated that a mutation in the AtCPSF73-II gene was detrimental to the genetic transmission of female gametes. Thus, two 73 kD subunits of the AtCPSF complex appear to have special functions during flower development. The important roles of mRNA 3′-end processing machinery in modulating plant development are discussed. Electronic supplementary material Electronic supplementary material is available for this article at and accessible for authorised users. Gene accession numbers associated with this paper: AY140902, AY140900, AY168923, AY140901     

A disulfide linkage in a CCCH zinc finger motif of an Arabidopsis CPSF30 ortholog

The Arabidopsis ortholog of the 30 kDa subunit of the cleavage and polyadenylation factor (AtCPSF30) is an RNA binding endonuclease, and the endonuclease activity is inhibited by reducing agents. Here, we report the presence of a disulfide linkage in the endonuclease motif based on comparative mass spectrometry (MS) analysis of reduced and non-reduced but carbamidomethylated protein. This analysis reveals that this disulfide bond involves a CCCH zinc finger motif, one that is associated with the endonuclease activity of AtCPSF30. This finding raises the possibility that redox regulation of AtCPSF30 may occur through oxidation and reduction of the disulfide linkage.     

Genetic identification of a second site modifier of ctr1-1 that controls ethylene-responsive and gravitropic root growth in Arabidopsis thaliana

Ethylene controls myriad aspects of plant growth throughout developmental stages in higher plants. It has been well established that ethylene-responsive growth entails extensive crosstalk with other plant hormones, particularly auxin. Here, we report a genetic mutation, named 1-aminocyclopropane carboxylic acid (ACC) resistant root1-1 (are1-1) in Arabidopsis thaliana (L.) Heynh. The CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) encodes a Raf-related protein, functioning as an upstream negative regulator of ethylene signaling in Arabidopsis thaliana. We found that the ctr1-1, a kinase-inactive allele exhibited slightly, but significantly, longer root length, compared to ACC-treated wild-type or ctr1-3, a null allele. Our genetic studies unveiled the existence of are1-1 mutation in the ctr1-1 mutant, as a second-site modifier which confers root-specific ethylene-resistance. Based on well-characterized crosstalk between ethylene and auxin during ethylene-responsive root growth, we performed various physiological analyses. Whereas are1-1 displayed normal sensitivity to synthetic auxins, it showed modest resistance to an auxin transport inhibitor, 1-Nnaphthylphthalamic acid. In addition, are1-1 mutant exhibited ectopically altered DR5:GUS activity upon ethylenetreatment. The results implicated the involvement of are1-1 in auxin-distribution, but not in auxin-biosynthesis, -uptake, or -sensitivity. In agreement, are1-1 mutant exhibited reduced gravitropic root growth and defective redistribution of DR5:GUS activity upon gravi-stimulation. Taken together with genetic and molecular analysis, our results suggest that ARE1 defines a novel locus to control ethylene-responsive root growth as well as gravitropic root growth presumably through auxin distribution in Arabidopsis thaliana.     

CIPK6, a CBL-interacting protein kinase is required for development and salt tolerance in plants

Calcineurin B-like proteins (CBL) and CBL-interacting protein kinases (CIPK) mediate plant responses to a variety of external stresses. Here we report that Arabidopsis CIPK6 is also required for the growth and development of plants. Phenotype of tobacco plants ectopically expressing a homologous gene ( CaCIPK6 ) from the leguminous plant chickpea ( Cicer arietinum ) indicated its functional conservation. A lesion in AtCIPK6 significantly reduced shoot-to-root and root basipetal auxin transport, and the plants exhibited developmental defects such as fused cotyledons, swollen hypocotyls and compromised lateral root formation, in conjunction with reduced expression of a number of genes involved in auxin transport and abiotic stress response. The Arabidopsis mutant was more sensitive to salt stress compared to wild-type, while overexpression of a constitutively active mutant of CaCIPK6 promoted salt tolerance in transgenic tobacco. Furthermore, tobacco seedlings expressing the constitutively active mutant of CaCIPK6 showed a developed root system, increased basipetal auxin transport and hypersensitivity to auxin. Our results provide evidence for involvement of a CIPK in auxin transport and consequently in root development, as well as in the salt-stress response, by regulating the expression of genes.