A plant SUMO ligase regulates the protein stability of a chromatin remodeling factor in root development.
Chromatin remodeling is essential for gene expression regulation in plant development and ...response to stresses. Brahma (BRM) is a conserved ATPase in the SWI/SNF chromatin remodeling complex and is involved in various biological processes in plant cells, but the regulation mechanism on BRM protein remains unclear. Here, we report that BRM interacts with AtMMS21, a SUMO ligase in Arabidopsis (
Arabidopsis thaliana
). The interaction was confirmed in different approaches in vivo and in vitro. The mutants of
BRM
and
AtMMS21
displayed a similar defect in root development. In the
mms21-1
mutant, the protein level of BRM-GFP was significantly lower than that in wild type, but the RNA level of
BRM
did not change. Biochemical evidence indicated that BRM was modified by SUMO3, and the reaction was enhanced by AtMMS21. Furthermore, overexpression of wild-type AtMMS21 but not the mutated AtMMS21 without SUMO ligase activity was able to recover the stability of BRM in
mms21-1
. Overexpression of
BRM
in
mms21-1
partially rescued the developmental defect of roots. Taken together, these results supported that AtMMS21 regulates the protein stability of BRM in root development.
In the transition from dark anaerobiosis to light, oxygen deactivates the hydrogenase pool, but only after carbon fixation outcompetes hydrogen production for electrons.
Photoproduction of hydrogen ...by green algae is considered a transitory release valve of excess reducing power and a potential carbon-free source of sustainable energy. It is generally accepted that the transitory production of hydrogen is governed by fast inactivation of hydrogenase by oxygen. However, our data suggest that photosynthetic electron loss to competing processes, mainly carbon fixation, stops hydrogen production, supports hydrogen uptake, and precedes the inevitable inactivation by oxygen. Here, we show that when transitioning from dark anaerobiosis to light, hydrogen production ceases within 2 min, regardless of the presence of oxygen. Simultaneous monitoring of the active hydrogenase pool size shows that it remains entirely intact up to 4 min after illumination and is inactivated only later. Thus, our data reveal a window of 4 min in which the hydrogenase pool is not being degraded by oxygen. Furthermore, we show that electron loss, prominently to carbon fixation, outcompetes hydrogen production and leads to hydrogen uptake. Indeed, supplying additional reducing power to hydrogenase at the cessation point regenerates the accumulation of hydrogen. Our results imply the fast cessation of hydrogen production is governed by electron loss rather than oxygen inactivation, which takes place minutes later.
Analysis of leaves from one angiosperm and four conifers where the mesophyll and vasculature can be mechanically separated revealed that the mesophyll produces substantial amounts of abscisic acid.
...The hormone abscisic acid (ABA) plays a critical role in enhancing plant survival during water deficit. Recent molecular evidence suggests that ABA is synthesized in the phloem companion cells and guard cells. However, the nature of cell turgor and water status in these two cell types cannot easily account for the rapid, water status-triggered ABA biosynthesis observed in leaves. Here, we utilize the unique foliar anatomies of an angiosperm (
Hakea lissosperma
) and four conifer species (
Saxegothaea conspicua
,
Podocarpus latifolius
,
Cephalotaxus harringtonii
, and
Amentotaxus formosana
) in which the mesophyll can be isolated from the vascular tissue to identify the main site of ABA biosynthesis in water-stressed leaves. In all five species tested, considerable ABA biosynthesis occurred in mesophyll tissue that had been separated from vascular tissue. In addition, the removal of the epidermis from the mesophyll in two conifer species had no impact on the observed increase in ABA levels under water deficit. Our results suggest that mesophyll cells are the predominant location of water deficit-triggered ABA biosynthesis in the leaf.
p-Coumarate acylates the γ-hydroxyls of lignin side chains, particularly on syringyl units, throughout all orders and various families of commelinid monocotyledons.
Commelinid monocotyledons are a ...monophyletic clade differentiated from other monocotyledons by the presence of cell wall-bound ferulate and
p
-coumarate. The Poaceae, or grass family, is a member of this group, and most of the
p
-coumarate in the cell walls of this family acylates lignin. Here, we isolated and examined lignified cell wall preparations from 10 species of commelinid monocotyledons from nine families other than Poaceae, including species from all four commelinid monocotyledon orders (Poales, Zingiberales, Commelinales, and Arecales). We showed that, as in the Poaceae, lignin-linked
p
-coumarate occurs exclusively on the hydroxyl group on the γ-carbon of lignin unit side chains, mostly on syringyl units. Although the mechanism of acylation has not been studied directly in these species, it is likely to be similar to that in the Poaceae and involve BAHD acyl-coenzyme A:monolignol transferases.
Complementation analysis reveals that C-terminal epitope tags have varied and unpredictable effects on FLS2 function.
Receptor-like kinases (RLKs) are the largest family of proteins in plants and are ...responsible for perceiving the vast majority of extracellular stimuli. Thus, RLKs function in diverse processes, including sensing pathogen attacks, regulating symbiotic interactions, transducing hormone and peptide signals, and monitoring cell wall status. However, despite their fundamental role in plant biology, very few antibodies are available against RLKs, which necessitates the use of epitope tags and fluorescent protein fusions in biochemical analyses such as immunoblot analysis and intracellular visualization. Epitope tags are widely used and are typically assumed to be benign, with no influence on protein function. FLAGELLIN SENSITIVE2 (FLS2) is the receptor for bacterial flagellin and often is used as a model for RLK function. Previous work implies that carboxyl-terminal epitope fusions to FLS2 maintain protein function. Here, a detailed complementation analysis of Arabidopsis (
Arabidopsis thaliana
)
fls2
mutant plants expressing various FLS2 C-terminal epitope fusions revealed highly variable and unpredictable FLS2-mediated signaling outputs. In addition, only one out of four FLS2 epitope fusions maintained the ability to inhibit plant growth in response to flg22 treatment comparable to that in the wild type or control untagged transgenic lines. These results raise concerns over the widespread use of RLK epitope tag fusions for functional studies. Many of the subtleties of FLS2 function, and by extension those of other RLKs, may have been overlooked or inappropriately interpreted through the use of RLK epitope tag fusions.
The C
4
species Gynandropsis gynandra exhibits natural variation in traits important for C
4
photosynthesis.
Engineering C
4
photosynthesis into C
3
crops could substantially increase their yield by ...alleviating photorespiratory losses. This objective is challenging because the C
4
pathway involves complex modifications to the biochemistry, cell biology, and anatomy of leaves. Forward genetics has provided limited insight into the mechanistic basis of these properties, and there have been no reports of significant quantitative intraspecific variation of C
4
attributes that would allow trait mapping. Here, we show that accessions of the C
4
species
Gynandropsis gynandra
collected from locations across Africa and Asia exhibit natural variation in key characteristics of C
4
photosynthesis. Variable traits include bundle sheath size and vein density, gas-exchange parameters, and carbon isotope discrimination associated with the C
4
state. The abundance of transcripts encoding core enzymes of the C
4
cycle also showed significant variation. Traits relating to water use showed more quantitative variation than those associated with carbon assimilation. We propose that variation in these traits likely adapted the hydraulic system for increased water use efficiency rather than improving carbon fixation, indicating that selection pressure may drive C
4
diversity in
G. gynandra
by modifying water use rather than photosynthesis. The accessions analyzed can be easily crossed and produce fertile offspring. Our findings, therefore, indicate that natural variation within this C
4
species is sufficiently large to allow genetic mapping of key C
4
traits and regulators.
The Arabidopsis (Arabidopsis thaliana) genome contains nine 𝛽-amylase (BAM) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized ...enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis BAM genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that BAM2 is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.
The gibberellin-induced increases in cytosolic Ca
2+
are promoted via a DELLA-independent signaling pathway.
DELLA proteins play a central role in gibberellin (GA) signaling. GA triggers DELLA ...degradation via the ubiquitin-proteasome pathway, thereby promoting plant growth. An increase in cytosolic Ca
2+
(Ca
2+
cyt
) was observed previously after several hours of GA application. Recent studies also suggest the existence of a DELLA-independent GA response. However, the effect of DELLA on the GA-induced increase in Ca
2+
cyt
remains unknown. This study reexamined the effects of GAs on Ca
2+
cyt
using the Ca
2+
sensor protein aequorin in Arabidopsis (
Arabidopsis thaliana
). Ca
2+
cyt
increased within a few minutes of GA treatment, even in transgenic plants expressing a mutated degradation-resistant version of REPRESSOR OF
ga1-3
and in
della
pentuple mutant plants. In addition, it was also revealed that Ca
2+
is not involved in DELLA degradation. These results suggest that the GA-induced increase in Ca
2+
cyt
occurs via a DELLA-independent pathway, providing important information on the GA signaling network.
An unusual β-amylase from Arabidopsis (BAM2) is catalytically active in the presence of K
+
, exhibits sigmoidal kinetics, functions as a tetramer, and has a putative secondary carbohydrate-binding ...site.
The Arabidopsis (
Arabidopsis thaliana
) genome contains nine β-amylase (
BAM
) genes, some of which play important roles in starch hydrolysis. However, little is known about BAM2, a plastid-localized enzyme reported to have extremely low catalytic activity. Using conservation of intron positions, we determined that the nine Arabidopsis
BAM
genes fall into two distinct subfamilies. A similar pattern was found in each major lineage of land plants, suggesting that these subfamilies diverged prior to the origin of land plants. Moreover, phylogenetic analysis indicated that
BAM2
is the ancestral member of one of these subfamilies. This finding, along with the conservation of amino acids in the active site of BAM2, suggested that it might be catalytically active. We then identified KCl as necessary for BAM2 activity. Unlike BAM1, BAM3, and BAM5, three Arabidopsis BAMs that all exhibited hyperbolic kinetics, BAM2 exhibited sigmoidal kinetics with a Hill coefficient of over 3. Using multi-angle light scattering, we determined that BAM2 was a tetramer, whereas BAM5 was a monomer. Conserved residues from a diverse set of BAM2 orthologs were mapped onto a homology model of the protein, revealing a large, conserved surface away from the active site that we hypothesize is a secondary carbohydrate-binding site. Introduction of bulky methionine for glycine at two points on this surface reduced catalytic activity significantly without disrupting the tetrameric structure. Expression analysis indicated that BAM2 is more closely coexpressed with other starch degradation enzymes than any other BAM, suggesting that BAM2 may play an important role in starch degradation in plants.
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