Members of the calcium-dependent protein kinase (CDPK/CPK) and SNF-related protein kinase (SnRK) superfamilies are commonly found in plants and some protists. Our knowledge of client specificity of the members of this superfamily is fragmentary. As this family is represented by over 30 members in Arabidopsis thaliana, the identification of kinase-specific and overlapping client relationships is crucial to our understanding the nuances of this large family of kinases as directed towards signal transduction pathways. Herein, we used the kinase client (KiC) assay—a relative, quantitative, high-throughput mass spectrometry-based in vitro phosphorylation assay—to identify and characterize potential CPK/SnRK targets of Arabidopsis. Eight CPKs (1, 3, 6, 8, 17, 24, 28, and 32), four SnRKs (subclass 1 and 2), and PPCK1 and PPCK2 were screened against a synthetic peptide library that contains 2095 peptides and 2661 known phosphorylation sites. A total of 625 in vitro phosphorylation sites corresponding to 203 non-redundant proteins were identified. The most promiscuous kinase, CPK17, had 105 candidate target proteins, many of which had already been discovered. Sequence analysis of the identified phosphopeptides revealed four motifs: LxRxxS, RxxSxxR, RxxS, and LxxxxS, that were significantly enriched among CPK/SnRK clients. The results provide insight into both CPK- and SnRK-specific and overlapping signaling network architectures and recapitulate many known in vivo relationships validating this large-scale approach towards discovering kinase targets.

The self-incompatibility (SI) system in the Solanaceae is comprised of cytotoxic pistil S-RNases which are countered by S-locus F-box (SLF) resistance factors found in pollen. Under this barrier-resistance architecture, mating system transitions from SI to self-compatibility (SC) typically result from loss-of-function mutations in genes encoding pistil SI factors such as S-RNase. However, the nature of these mutations is often not well characterized. Here we use a combination of S-RNase sequence analysis, transcript profiling, protein expression and reproductive phenotyping to better understand different mechanisms that result in loss of S-RNase function. Our analysis focuses on 12 S-RNase alleles identified in SC species and populations across the tomato clade. In six cases, the reason for gene dysfunction due to mutations is evident. The six other alleles potentially encode functional S-RNase proteins but are typically transcriptionally silenced. We identified three S-RNase alleles which are transcriptionally silenced under some conditions but actively expressed in others. In one case, expression of the S-RNase is associated with SI. In another case, S-RNase expression does not lead to SI, but instead confers a reproductive barrier against pollen tubes from other tomato species. In the third case, expression of S-RNase does not affect self, interspecific or inter-population reproductive barriers. Our results indicate that S-RNase expression is more dynamic than previously thought, and that changes in expression can impact different reproductive barriers within or between natural populations.

Tomato clade species (Solanum sect. Lycopersicon) display multiple interspecific reproductive barriers (IRBs). Some IRBs conform to the SI x SC rule, which describes unilateral incompatibility (UI) where pollen from SC species is rejected on SI species’ pistils, but reciprocal pollinations are successful. However, SC x SC UI also exists, offering opportunities to identify factors that contribute to S-RNase-independent IRBs. For instance, SC Solanum pennellii LA0716 pistils only permit SC Solanum lycopersicum pollen tubes to penetrate to the top third of the pistil, while S. pennellii pollen penetrates to S. lycopersicum ovaries. We identified candidate S. pennellii LA0716 pistil barrier genes based on expression profiles and published results. CRISPR/Cas9 mutants were created in eight candidate genes, and mutants were assessed for changes in S. lycopersicum pollen tube growth. Mutants in a gene designated Defective in Induced Resistance 1-like (SpDIR1L), which encodes a small cysteine-rich protein, permitted S. lycopersicum pollen tubes to grow to the bottom third of the style. We show that SpDIR1L protein accumulation correlates with IRB strength and that species with weak or no IRBs toward S. lycopersicum pollen share a 150 bp deletion in the upstream region of SpDIR1L. These results suggest that SpDIR1L contributes to an S-RNase-independent IRB.

Mating system transitions from self-incompatibility (SI) to self-compatibility (SC) are common in plants. In the absence of high levels of inbreeding depression, SC alleles are predicted to spread due to transmission advantage and reproductive assurance. We characterized mating system and pistil-expressed SI factors in 20 populations of the wild tomato species Solanum habrochaites from the southern half of the species range. We found that a single SI to SC transition is fixed in populations south of the Rio Chillon valley in central Peru. In these populations, SC correlated with the presence of the hab-6 S-haplotype that encodes a low activity S-RNase protein. We identified a single population segregating for SI/SC and hab-6. Intrapopulation crosses showed that hab-6 typically acts in the expected codominant fashion to confer SC. However, we found one specific S-haplotype (hab-10) that consistently rejects pollen of the hab-6 haplotype, and results in SI hab-6/hab-10 heterozygotes. We suggest that the hab-10 haplotype could act as a genetic mechanism to stabilize mixed mating in this population by presenting a disadvantage for the hab-6 haplotype. This barrier may represent a mechanism allowing for the persistence of SI when an SC haplotype appears in or invades a population.

A goal of speciation genetics is to understand how the genetic components underlying interspecific reproductive barriers originate within species. Unilateral incompatibility (UI) is a postmating prezygotic barrier in which pollen rejection in the female reproductive tract (style) occurs in only one direction of an interspecific cross. Natural variation in the strength of UI has been observed among populations within species in the wild tomato clade. In some cases, molecular loci underlying self-incompatibility (SI) are associated with this variation in UI, but the mechanistic connection between these intra- and inter-specific pollen rejection behaviors is poorly understood in most instances. We generated an F-2 population between SI and SC genotypes of a single species, Solanum pennellii, to examine the genetic basis of intraspecific variation in UI against other species, and to determine whether loci underlying SI are genetically associated with this variation. We found that F-2 individuals vary in the rate at which UI rejection occurs. One large effect QTL detected for this trait co-localized with the SI-determining S-locus. Moreover, individuals that expressed S-RNase-the S-locus protein involved in SI pollen rejection-in their styles had much more rapid UI responses compared with those without S-RNase protein. Our analysis shows that intraspecific variation at mate choice loci-in this case at loci that prevent self-fertilization-can contribute to variation in the expression of interspecific isolation, including postmating prezygotic barriers. Understanding the nature of such intraspecific variation can provide insight into the accumulation of these barriers between diverging lineages.

In plants, transitions in mating system from outcrossing to self-fertilization are common; however, the impact of these transitions on interspecific and interpopulation reproductive barriers is not fully understood. We examined the consequences of mating system transition for reproductive barriers in 19 populations of the wild tomato species Solanum habrochaites. We identified S. habrochaites populations with self-incompatible (SI), self-compatible (SC) and mixed population (MP) mating systems, and characterized pollen-pistil interactions among S. habrochaites populations and between S. habrochaites and other tomato species. We examined the relationship between mating system, floral morphology, interspecific and interpopulation compatibility and pistil SI factors. We documented five distinct phenotypic groups by combining reproductive behavior with molecular data. Transitions from SI to MP were not associated with weakened interspecific reproductive barriers or loss of known pistil SI factors. However, transitions to SC at the northern range margin were accompanied by loss of S-RNase, smaller flowers, and weakened (or absent) interspecific pollen-pistil barriers. Finally, we identified a subset of SC populations that exhibited a partial interpopulation reproductive barrier with central SI populations. Our results support the hypothesis that shifts in mating system, followed by additional loss-of-function mutations, impact reproductive barriers within and between species.

Plants have mechanisms to recognize and reject pollen from other species. Although widespread, these mechanisms are less well understood than the self-incompatibility (SI) mechanisms plants use to reject pollen from close relatives. Previous studies have shown that some interspecific reproductive barriers (IRBs) are related to SI in the Solanaceae. For example, the pistil SI proteins S-RNase and HT protein function in a pistil-side IRB that causes rejection of pollen from self-compatible (SC) red/orange-fruited species in the tomato clade. However, S-RNase-independent IRBs also clearly contribute to rejecting pollen from these species. We investigated S-RNase-independent rejection of Solanum lycopersicum pollen by SC Solanum pennellii LA0716, SC. Solanum habrochaites LA0407, and SC Solanum arcanum LA2157, which lack functional S-RNase expression. We found that all three accessions express HT proteins, which previously had been known to function only in conjunction with S-RNase, and then used RNAi to test whether they also function in S-RNase-independent pollen rejection. Suppressing HT expression in SC S. pennellii LA0716 allows S. lycopersicum pollen tubes to penetrate farther into the pistil in HT suppressed plants, but not to reach the ovary. In contrast, suppressing HT expression in SC. Solanum habrochaites LA0407 and in SC S. arcanum LA2157 allows S. lycopersicum pollen tubes to penetrate to the ovary and produce hybrids that, otherwise, would be difficult to obtain. Thus, HT proteins are implicated in both S-RNase-dependent and S-RNase-independent pollen rejection. The results support the view that overall compatibility results from multiple pollen-pistil interactions with additive effects.

PREMISE OF THE STUDY:Self-incompatibility (SI) is a mechanism that prevents inbreeding in many plant species. The mutational breakdown of SI occurs frequently, yet relatively little is known about the evolutionary steps involved in the progressive loss of pistil and pollen SI function.METHODS:In Solanaceae, SI is the S-RNase-based gametophytic type. We used SI and SC populations of the wild tomato species Solanum habrochaites to study natural variation for two pollen SI factors: a Cullin1 (CUL1) protein and an S-locus F-box protein (SLF-23). Pollen compatibility was assessed on an allotriploid tester line encoding an S-RNase recognized by SLF-23. Both pollen factors are required for compatibility on this tester line. Complementation tests and gene sequencing were used to identify mutations in CUL1 or SLF-23.KEY RESULTS:We detected loss-of-function mutations in CUL1 and/or SLF-23 in SC populations collected near the northern and southern geographic margins of this taxon's natural range. Nonmarginal SC and all SI accessions expressed mostly functional alleles of these pollen factors. Comparison of the CUL1 sequences identified several shared deletion mutations present in both northern and southern margin SC accessions.CONCLUSIONS:Loss-of-function mutations in CUL1 and SLF-23 likely became fixed relatively late during SI to SC transitions, after loss of pistil SI function. Mutations in CUL1 establish unilateral incompatibility with SI populations and strengthen reproductive isolation. Point mutations common to northern and southern SC biotypes likely derive from shared ancestral variants found in more central SI populations.

Pollen-pistil interactions contribute to mate selection at the postmating, prezygotic level.

In a new study, the Papaver rhoeas (poppy family) self-incompatibility system has been transferred into Arabidopsis thaliana, a distantly related plant with a very different floral structure. The simple poppy self-incompatibility system may finally make it possible to introduce this potentially valuable trait into any plant.

Interspecific reproductive barriers are poorly understood, but are central to the biological species concept. The pre-zygotic barriers between red- and green-fruited species in the tomato clade of the genus Solanum provide a model to better understand these barriers in plants. Compatibility usually follows the SI x SC rule: pollen from self-compatible (SC) red-fruited species is rejected on pistils of the predominantly self-incompatible (SI) green-fruited species, but the reciprocal crosses are compatible. This suggests that the interspecific reproductive barrier may be linked to the intraspecific SI mechanism. However, pollen from the SC red-fruited species is also rejected by SC accessions of green-fruited species that lack S-RNase, a key protein expressed in pistils of SI Solanum species. Thus, multiple mechanisms may contribute to the barrier between red- and green-fruited species. We tested whether an S-RNase-dependent barrier is sufficient for rejection of pollen from red-fruited species by introducing functional S-RNase, HT-A and HT-B genes from SI species into Solanum lycopersicum (cultivated tomato). We found that expressing S-RNase in combination with either HT-A or HT-B in the pistil is sufficient to cause rejection of pollen from all four red-fruited species. Thus, redundant mechanisms must operate side by side to prevent crosses between red- and green-fruited species in the clade, underlining the complexity of interspecific pollination barriers. Our results also have implications for mating system transitions. We suggest that these transitions must occur in a specific sequence, and that the transition from SI to SC also affects interspecific compatibility.

The Arabidopsis thaliana genome includes three genes for mitochondrial dihydrolipoamide acetyltransferase, the E2-component of the mitochondrial pyruvate dehydrogenase complex (PDC). Two genes encode E2-proteins with a single lipoyl domain, while the third has a two-lipoyl domain structure. Transcripts for each E2 protein were expressed in all plant organs. Each recombinant AtmtE2 can individually form an icosahedral PDC core structure, and results from bimolecular fluorescence complementation assays are consistent with formation of hetero-core structures from all permutations of the AtmtE2 proteins. We propose a unique regulatory mechanism involving dynamic formation of hetero-core complexes that include both mono- and di-lipoyl forms of AtmtE2.

In the reference dicot plant Arabidopsis thaliana , the PP2C family of P-protein phosphatases includes the products of 80 genes that have been separated into ten multi-protein clades plus six singletons. Clade D includes the products of nine genes distributed among three chromosomes (APD1, At3g12620; APD2, At3g17090; APD3, At3g51370; APD4, At3g55050; APD5, At4g33920; APD6, At4g38520; APD7, At5g02760; APD8, At5g06750; and APD9, At5g66080). As part of a functional genomics analysis of protein phosphorylation, we retrieved expression data from public databases and determined the subcellular protein localization of the members of clade D. While the nine proteins have been grouped together based upon primary sequence alignments, we observed no obvious common patterns in expression or localization. We found chimera with the GFP associated with the nucleus, plasma membrane, the endomembrane system, and mitochondria in transgenic plants.

While more than a thousand protein kinases (PK) have been identified in the Arabidopsis thaliana genome, relatively little progress has been made toward identifying their individual client proteins. Herein we describe the use of a mass spectrometry-based in vitro phosphorylation strategy, termed Kinase Client assay (KiC assay), to study a targeted-aspect of signaling. A synthetic peptide library comprising 377 in vivo phosphorylation sequences from developing seed was screened using 71 recombinant A. thaliana PK. Among the initial results, we identified 23 proteins as putative clients of 17 PK. In one instance protein phosphatase inhibitor-2 (AtPPI-2) was phosphorylated at multiple-sites by three distinct PK, casein kinase1-like 10, AME3, and a Ser PK-like protein. To confirm this result, full-length recombinant AtPPI-2 was reconstituted with each of these PK. The results confirmed multiple distinct phosphorylation sites within this protein. Biochemical analyses indicate that AtPPI-2 inhibits type 1 protein phosphatase (TOPP) activity, and that the phosphorylated forms of AtPPI-2 are more potent inhibitors. Structural modeling revealed that phosphorylation of AtPPI-2 induces conformational changes that modulate TOPP binding.

Although self-incompatibility (SI) in plants has been studied extensively, far less is known about interspecific reproductive barriers. One interspecific barrier, known as unilateral incongruity or incompatibility (UI), occurs when species display unidirectional compatibility in interspecific crosses. In the wild tomato species Solanum pennellii, both SI and self-compatible (SC) populations express UI when crossed with domesticated tomato, offering a useful model system to dissect the molecular mechanisms involved in reproductive barriers. In this study, the timing of reproductive barrier establishment during pistil development was determined in SI and SC accessions of S. pennellii using a semi-in vivo system to track pollen-tube growth in developing styles. Both SI and UI barriers were absent in styles 5 days prior to flower opening, but were established by 2 days before flower opening, with partial barriers detected during a transition period 34 days before flower opening. The developmental expression dynamics of known SI factors, S-RNases and HT proteins, was also examined. The accumulation of HT-A protein coincided temporally and spatially with UI barriers in developing pistils. Proteomic analysis of stigma/styles from key developmental stages showed a switch in protein profiles from cell-division-associated proteins in immature stigma/styles to a set of proteins in mature stigma/styles that included S-RNases, HT-A protein and proteins associated with cell-wall loosening and defense responses, which could be involved in pollenpistil interactions. Other prominent proteins in mature stigma/styles were those involved in lipid metabolism, consistent with the accumulation of lipid-rich material during pistil maturation.

Peroxiredoxins (Prxs), thioredoxins (Trxs), and NADPH-thioredoxin reductases (NTRs) constitute central elements of the thiol-disulfide redox regulatory network of plant cells. This study provides a comprehensive survey of this network in the model legume Lotus japonicus. The aims were to identify and characterize these gene families and to assess whether the NTR-Trx systems are operative in nodules. Quantitative reverse transcription-polymerase chain reaction and immunological and proteomic approaches were used for expression profiling. We identified seven Prx, 14 Trx, and three NTR functional genes. The PrxQ1 gene was found to be transcribed in two alternative spliced variants and to be expressed at high levels in leaves, stems, petals, pods, and seeds and at low levels in roots and nodules. The 1CPrx gene showed very high expression in the seed embryos and low expression in vegetative tissues and was induced by nitric oxide and cytokinins. In sharp contrast, cytokinins down-regulated all other Prx genes, except PrxQ1, in roots and nodules, but only 2CPrxA and PrxQ1 in leaves. Gene-specific changes in Prx expression were also observed in response to ethylene, abscisic acid, and auxins. Nodules contain significant mRNA and protein amounts of cytosolic PrxIIB, Trxh1, and NTRA and of plastidic NTRC. Likewise, they express cytosolic Trxh3, Trxh4, Trxh8, and Trxh9, mitochondrial PrxIIF and Trxo, and plastidic Trxm2, Trxm4, and ferredoxin-Trx reductase. These findings reveal a complex regulation of Prxs that is dependent on the isoform, tissue, and signaling molecule and support that redox NTR-Trx systems are functional in the cytosol, mitochondria, and plastids of nodules.

We have developed anin vitrosystem for detailed analysis of reversible phosphorylation of the plant mitochondrial pyruvate dehydrogenase complex, comprising recombinantArabidopsis thalianaα2β2-heterotetrameric pyruvate dehydrogenase (E1) plusA. thalianaE1-kinase (AtPDK). Upon addition of MgATP, Ser292, which is located within the active-site loop structure of E1α, is phosphorylated. In addition to Ser292, Asp295 and Gly297 are highly conserved in the E1α active-site loop sequences. Mutation of Asp295 to Ala, Asn, or Leu greatly reduced phosphorylation of Ser292, while mutation of Gly297 had relatively little effect. Quantitative two-hybrid analysis was used to show that mutation of Asp295 did not substantially affect binding of AtPDK to E1α. When using pyruvate as a variable substrate, the Asp295 mutant proteins had modest changes inkcat,Km, andkcat/Kmvalues. Therefore, we propose that Asp295 plays an important role in stabilizing the active-site loop structure, facilitating transfer of the γ-phosphate from ATP to the Ser residue at regulatory site one of E1α.