Rubisco activase is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of Rubisco during photosynthesis. In Arabidopsis, Rubisco activase is negatively regulated by dark-dependent phosphorylation of Thr78. The prevalence of Thr78 in Rubisco activase was investigated across sequences from 91 plant species, finding that 29 (∼32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a Thr78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of Thr78 was observed only in Arabidopsis. Further, phosphorylation of Thr78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of Thr78 in Rubisco activase from a wide range of species, a regulatory role for phosphorylation at this site is more limited. This provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein.

Rubisco activase (Rca) is an ATP-dependent chaperone that facilitates dissociation of inhibitory sugar phosphates from the catalytic sites of ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) during photosynthesis. In Arabidopsis, Rca is negatively regulated by dark-dependent phosphorylation of Thr78. Here we identify chloroplast localized SHEWENELLA-LIKE PROTEIN PHOSPHATASE 1 (SLP1) as the major phosphatase responsible for light-dependent Rca dephosphorylation. The prevalence of Thr78 in Rca was investigated across sequences from 91 plant species, finding 29 (~32%) species shared a threonine in the same position. Analysis of seven C3 species with an antibody raised against a Thr78 phospho-peptide demonstrated that this position is phosphorylated in multiple genera. However, light-dependent dephosphorylation of Thr78 was observed only in Arabidopsis. Further, phosphorylation of Thr78 could not be detected in any of the four C4 grass species examined. The results suggest that despite conservation of Thr78 in Rca from a wide range of species, a regulatory role for phosphorylation at this site may be limited. This provides a case study for how variation in post-translational regulation can amplify functional divergence across the phylogeny of plants beyond what is explained by sequence variation in a metabolically important protein. ### Competing Interest Statement The authors have declared no competing interest.

Rubisco activase (Rca) facilitates the catalytic repair of Rubisco, the CO 2 -fixing enzyme of photosynthesis, following periods of darkness, low to high light transitions or stress. Removal of the redox-regulated isoform of Rubisco activase, Rca-α, enhances photosynthetic induction in Arabidopsis and has been suggested as a strategy for the improvement of crops, which may experience frequent light transitions in the field; however, this has never been tested in a crop species. Therefore, we used RNAi to reduce the Rca-α content of soybean ( Glycine max cv. Williams 82) below detectable levels and then characterized the growth, photosynthesis, and Rubisco activity of the resulting transgenics, in both growth chamber and field conditions. Under a 16 h sine wave photoperiod, the reduction of Rca-α contents had no impact on morphological characteristics, leaf expansion rate, or total biomass. Photosynthetic induction rates were unaltered in both chamber-grown and field-grown plants. Plants with reduced Rca-α content maintained the ability to regulate Rubisco activity in low light just as in control plants. This result suggests that in soybean, Rca-α is not as centrally involved in the regulation of Rca oligomer activity as it is in Arabidopsis . The isoform stoichiometry supports this conclusion, as Rca-α comprises only ~ 10% of the Rubisco activase content of soybean, compared to ~ 50% in Arabidopsis . This is likely to hold true in other species that contain a low ratio of Rca-α to Rca-ß isoforms.

Rubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors at Rubisco catalytic sites during CO 2 fixation. Most plant species express two Rca isoforms, the larger Rca‐α and the shorter Rca‐β, either by alternative splicing from a single gene or expression from separate genes. The mechanism of Rubisco activation by Rca isoforms has been intensively studied in C 3 plants. However, the functional role of Rca in C 4 plants where Rubisco and Rca are located in a much higher [CO 2 ] compartment is less clear. In this study, we selected four C 4 bioenergy grasses and the model C 4 grass setaria ( Setaria viridis ) to investigate the role of Rca in C 4 photosynthesis. All five C 4 grass species contained two Rca genes, one encoding Rca‐α and the other Rca‐β, which were positioned closely together in the genomes. A variety of abiotic stress‐related motifs were identified in the Rca‐α promoter of each grass, and while the Rca‐β gene was constantly highly expressed at ambient temperature, Rca‐α isoforms were expressed only at high temperature but never surpassed 30% of Rca‐β content. The pattern of Rca‐α induction on transition to high temperature and reduction on return to ambient temperature was the same in all five C 4 grasses. In sorghum ( Sorghum bicolor ), sugarcane ( Saccharum officinarum ), and setaria, the induction rate of Rca‐α was similar to the recovery rate of photosynthesis and Rubisco activation at high temperature. This association between Rca‐α isoform expression and maintenance of Rubisco activation at high temperature suggests that Rca‐α has a functional thermo‐protective role in carbon fixation in C 4 grasses by sustaining Rubisco activation at high temperature.

Transposable elements (TEs) are a ubiquitous feature of plant genomes. Because of the threat they post to genome integrity, most TEs are epigenetically silenced. However, even closely related plant species often have dramatically different populations of TEs, suggesting periodic rounds of activity and silencing. Here, we show that the process of de novo methylation of an active element in maize involves two distinct pathways, one of which is directly implicated in causing epigenetic silencing and one of which is the result of that silencing. Epigenetic changes involve changes in gene expression that can be heritably transmitted to daughter cells in the absence of changes in DNA sequence. Epigenetics has been implicated in phenomena as diverse as development, stress response, and carcinogenesis. A significant challenge facing those interested in investigating epigenetic phenomena is determining causal relationships between DNA methylation, specific classes of small RNAs, and associated changes in gene expression. Because they are the primary targets of epigenetic silencing in plants and, when active, are often targeted for de novo silencing, TEs represent a valuable source of information about these relationships. We use a naturally occurring system in which a single TE can be heritably silenced by a single derivative of that TE. By using this system it is possible to unravel causal relationships between different size classes of small RNAs, patterns of DNA methylation, and heritable silencing. Here, we show that the long terminal inverted repeats within Zea mays MuDR transposons are targeted by distinct classes of small RNAs during epigenetic silencing that are dependent on distinct silencing pathways, only one of which is associated with transcriptional silencing of the transposon. Further, these small RNAs target distinct regions of the terminal inverted repeats, resulting in different patterns of cytosine methylation with different functional consequences with respect to epigenetic silencing and the heritability of that silencing.

46 Epigenetic changes involve changes in gene expression that can be heritably transmitted 47 to daughter cells in the absence of changes in DNA sequence. Epigenetics has been 48 implicated in phenomena as diverse as development, stress response and carcinogenesis. 49 A significant challenge facing those interested in investigating epigenetic phenomena is 50 determining causal relationships between DNA methylation, specific classes of small 51 RNAs and associated changes in gene expression. Because they are the primary targets 52 of epigenetic silencing in plants and, when active, are often targeted for de novo 53 silencing, transposable elements (TEs) represent a valuable source of information about 54 these relationships. We use a naturally occurring system in which a single TE can be 55 heritably silenced by a single derivative of that TE. By using this system it is possible to 56 unravel causal relationships between different size classes of small RNAs, patterns of 57 DNA methylation and heritable silencing. Here, we show that the long terminal inverted 58 repeats (TIRs) within Zea mays MuDR transposons are targeted by distinct classes of 59

SUMMARYRubisco activase (Rca) facilitates the release of sugar‐phosphate inhibitors from the active sites of Rubisco and thereby plays a central role in initiating and sustaining Rubisco activation. In Arabidopsis, alternative splicing of a single Rca gene results in two Rca isoforms, Rca‐α and Rca‐β. Redox modulation of Rca‐α regulates the function of Rca‐α and Rca‐β acting together to control Rubisco activation. Although Arabidopsis Rca‐α alone less effectively activates Rubisco in vitro, it is not known how CO2 assimilation and plant growth are impacted. Here, we show that two independent transgenic Arabidopsis lines expressing Rca‐α in the absence of Rca‐β (‘Rca‐α only’ lines) grew more slowly in various light conditions, especially under low light or fluctuating light intensity, and in a short day photoperiod compared to wildtype. Photosynthetic induction was slower in the Rca‐α only lines, and they maintained a lower rate of CO2 assimilation during both photoperiod types. Our findings suggest Rca oligomers composed of Rca‐α only are less effective in initiating and sustaining the activation of Rubisco than when Rca‐β is also present. Currently there are no examples of any plant species that naturally express Rca‐α only but numerous examples of species expressing Rca‐β only. That Rca‐α exists in most plant species, including many C3 and C4 food and bioenergy crops, implies its presence is adaptive under some circumstances.

Seed dormancy is a critical mechanism that delays germination until environmental conditions are favorable for growth. Plant hormones gibberellin (GA) and abscisic acid (ABA) have long been recognized as key players in regulating dormancy and germination. Recent data have increased interest in brassinosteroid (BR) hormones that promote germination by activating GA downstream genes and inactivating ABA signaling. Exposure of imbibed seeds to low temperature (cold stratification) is widely used to release seed dormancy and to improve germination frequency. However, the mechanism by which cold stratification overcomes the inhibitory role of ABA is not completely understood. In the present study, we show delayed germination of seeds of the BR insensitive mutant, bri1-5, that was largely reversed by treatment with fluridone, an inhibitor of ABA biosynthesis. In addition, the bri1-5 seeds were markedly less sensitive to the cold stratification release of dormancy. These results suggest that BR locates upstream of ABA signaling and downstream of cold stratification signaling in dormancy and germination pathways. Consistent with this notion, BR biosynthetic genes, DWF4 and DET2, were upregulated by cold stratification. The transcripts of the GA biosynthesis gene, GA3ox1, and cold responsive genes, CBF1 and CBF2, increased in response to cold stratification in wild type seeds but not in bri1-5 seeds. Conversely, transgenic seeds overexpressing BRI1 germinated more rapidly than wild type in the absence of cold stratification. Thus, we propose that BR signaling plays a previously unrecognized role in the cold stratification pathway for seed dormancy and germination.

Arabidopsis Rubisco activase (Rca) is phosphorylated at threonine-78 (Thr78) in low light and in the dark, suggesting a potential regulatory role in photosynthesis, but this has not been directly tested. To do so, we transformed an rca -knockdown mutant largely lacking redox regulation with wild-type Rca-β or Rca-β with Thr78-to-Ala (T78A) or Thr78-to-Ser (T78S) site–directed mutations. Interestingly, the T78S mutant was hyperphosphorylated at the Ser78 site relative to Thr78 of the Rca-β wild-type control, as evidenced by immunoblotting with custom antibodies and quantitative mass spectrometry. Moreover, plants expressing the T78S mutation had reduced photosynthesis and quantum efficiency of photosystem II (ϕ PSII ) and reduced growth relative to control plants expressing wild-type Rca-β under all conditions tested. Gene expression was also altered in a manner consistent with reduced growth. In contrast, plants expressing Rca-β with the phospho-null T78A mutation had faster photosynthetic induction kinetics and increased ϕ PSII relative to Rca-β controls. While expression of the wild-type Rca-β or the T78A mutant fully rescued the slow-growth phenotype of the rca- knockdown mutant grown in a square-wave light regime, the T78A mutants grew faster than the Rca-β control plants at low light (30 µmol photons m −2 s −1 ) and in a fluctuating low-light/high-light environment. Collectively, these results suggest that phosphorylation of Thr78 (or Ser78 in the T78S mutant) plays a negative regulatory role in vivo and provides an explanation for the absence of Ser at position 78 in terrestrial plant species.

Binding of brassinolide to the BRASSINOSTEROID-INSENSTIVE 1 (BRI1) receptor kinase promotes interaction with its co-receptor, BRI1-ASSOCIATED RECEPTOR KINASE 1 (BAK1). Juxtaposition of the kinase domains that occurs then allows reciprocal transphosphorylation and activation of both kinases, but details of that process are not entirely clear. In the present study we show that the carboxy (C) - terminal polypeptide of BAK1 may play a role. First, we demonstrate that the C-terminal domain is a strong inhibitor of the transphosphorylation activity of the recombinant BAK1 cytoplasmic domain protein. However, recombinant BAK1 lacking the C-terminal domain is unable to transactivate the peptide kinase activity of BRI1 in vitro. Thus, the C-terminal domain may play both a positive and negative role. Interestingly, a synthetic peptide corresponding to the full C-terminal domain (residues 576 to 615 of BAK1) interacted with recombinant BRI1 in vitro, and that interaction was enhanced by phosphorylation at the Tyr-610 site. Expression of a BAK1 C-terminal domain truncation (designated BAK1-ΔCT-Flag) in transgenic Arabidopsis plants lacking endogenous bak1 and its functional paralog, bkk1, produced plants that were wild type in appearance but much smaller than plants expressing full-length BAK1-Flag. The reduction in growth may be attributed to a partial inhibition of BR signaling in vivo as reflected in root growth assays but other factors are likely involved as well. Our working model is that in vivo, the inhibitory action of the C-terminal domain of BAK1 is relieved by binding to BRI1. However, that interaction is not essential for BR signaling, but other aspects of cellular signaling are impacted when the C-terminal domain is truncated and result in inhibition of growth. These results increase the molecular understanding of the C-terminal domain of BAK1 as a regulator of kinase activity that may serve as a model for other receptor kinases.