Arabidopsis Raf‐like kinases act as positive regulators of subclass III SnRK2 in osmostress signaling

Given their sessile nature, land plants must use various mechanisms to manage dehydration under water‐deficit conditions. Osmostress‐induced activation of the SNF1‐related protein kinase 2 (SnRK2) family elicits physiological responses such as stomatal closure to protect plants during drought conditions. With the plant hormone ABA receptors [PYR (pyrabactin resistance)/PYL (pyrabactin resistance‐like)/RCAR (regulatory component of ABA receptors) proteins] and group A protein phosphatases, subclass III SnRK2 also constitutes a core signaling module for ABA, and osmostress triggers ABA accumulation. How SnRK2 is activated through ABA has been clarified, although its activation through osmostress remains unclear. Here, we show that Arabidopsis ABA and abiotic stress‐responsive Raf‐like kinases (AtARKs) of the B3 clade of the mitogen‐activated kinase kinase kinase (MAPKKK) family are crucial in SnRK2‐mediated osmostress responses. Disruption of AtARKs in Arabidopsis results in increased water loss from detached leaves because of impaired stomatal closure in response to osmostress. Our findings obtained in vitro and in planta have shown that AtARKs interact physically with SRK2E, a core factor for stomatal closure in response to drought. Furthermore, we show that AtARK phosphorylates S171 and S175 in the activation loop of SRK2E in vitro and that Atark mutants have defects in osmostress‐induced subclass III SnRK2 activity. Our findings identify a specific type of B3‐MAPKKKs as upstream kinases of subclass III SnRK2 in Arabidopsis. Taken together with earlier reports that ARK is an upstream kinase of SnRK2 in moss, an existing member of a basal land plant lineage, we propose that ARK/SnRK2 module is evolutionarily conserved across 400 million years of land plant evolution for conferring protection against drought.     

LCI1, a Chlamydomonas reinhardtii plasma membrane protein,functions in active CO2 uptake under low CO2

In response to high CO₂ environmental variability, green algae, such as Chlamydomonas reinhardtii, have evolved multiple physiological states dictated by external CO₂ concentration. Genetic and physiological studies demonstrated that at least three CO₂ physiological states, a high CO₂ (0.5–5% CO₂), a low CO₂ (0.03–0.4% CO₂) and a very low CO₂ (< 0.02% CO₂) state, exist in Chlamydomonas. To acclimate in the low and very low CO₂ states, Chlamydomonas induces a sophisticated strategy known as a CO₂‐concentrating mechanism (CCM) that enables proliferation and survival in these unfavorable CO₂ environments. Active uptake of C_i from the environment is a fundamental aspect in the Chlamydomonas CCM, and consists of CO₂ and HCO₃^– uptake systems that play distinct roles in low and very low CO₂ acclimation states. LCI1, a putative plasma membrane C_i transporter, has been linked through conditional overexpression to active C_i uptake. However, both the role of LCI1 in various CO₂ acclimation states and the species of C_i, HCO₃^– or CO₂, that LCI1 transports remain obscure. Here we report the impact of an LCI1 loss‐of‐function mutant on growth and photosynthesis in different genetic backgrounds at multiple pH values. These studies show that LCI1 appears to be associated with active CO₂ uptake in low CO₂, especially above air‐level CO₂, and that any LCI1 role in very low CO₂ is minimal.     

Extraordinarily conserved chromosomal synteny of Citrus species revealed by chromosome‐specific painting

Reliable identification of individual chromosomes in eukaryotic species is the foundation for comparative chromosome synteny and evolutionary studies. Unfortunately, chromosome identification has been a major challenge for plants with small chromosomes, such as the Citrus species. We developed oligonucleotide‐based chromosome painting probes for all nine chromosomes in Citrus maxima (Pummelo). We were able to identify all C. maxima chromosomes in the same metaphase cells using multiple rounds of sequential fluorescence in situ hybridization with the painting probes. We conducted comparative chromosome painting analysis in six different Citrus and related species. We found that each painting probe hybridized to only a single chromosome in all other five species, suggesting that the six species have maintained a complete chromosomal synteny after more than 9 million years of divergence. No interchromosomal rearrangement was identified in any species. These results support the hypothesis that karyotypes of woody species are more stable than herbaceous plants because woody plants need a longer period to fix chromosome structural variants in natural populations.     

Model‐assisted identification of metabolic engineering strategies for Jatropha curcas lipid pathways

Efficient approaches to increase plant lipid production are necessary to meet current industrial demands for this important resource. While Jatropha curcas cell culture can be used for in vitro lipid production, scaling up the system for industrial applications requires an understanding of how growth conditions affect lipid metabolism and yield. Here we present a bottom‐up metabolic reconstruction of J. curcas supported with labeling experiments and biomass characterization under three growth conditions. We show that the metabolic model can accurately predict growth and distribution of fluxes in cell cultures and use these findings to pinpoint energy expenditures that affect lipid biosynthesis and metabolism. In addition, by using constraint‐based modeling approaches we identify network reactions whose joint manipulation optimizes lipid production. The proposed model and computational analyses provide a stepping stone for future rational optimization of other agronomically relevant traits in J. curcas.     

Expansion of MIR482/2118 by a class‐II transposable element in cotton

Some plant microRNA (miRNA) families contain multiple members generating identical or highly similar mature miRNA variants. Mechanisms underlying the expansion of miRNA families remain elusive, although tandem and/or segmental duplications have been proposed. In this study of two tetraploid cottons, Gossypium hirsutum and Gossypium barbadense, and their extant diploid progenitors, Gossypium arboreum and Gossypium raimondii, we investigated the gain and loss of members of the miR482/2118 superfamily, which modulates the expression of nucleotide‐binding site leucine‐rich repeat (NBS‐LRR) disease resistance genes. We found significant expansion of MIR482/2118d in G. barbadense, G. hirsutum and G. raimondii, but not in G. arboreum. Several newly expanded MIR482/2118d loci have mutated to produce different miR482/2118 variants with altered target‐gene specificity. Based on detailed analysis of sequences flanking these MIR482/2118 loci, we found that this expansion of MIR482/2118d and its derivatives resulted from an initial capture of an MIR482/2118d by a class‐II DNA transposable element (TE) in G. raimondii prior to the tetraploidization event, followed by transposition to new genomic locations in G. barbadense, G. hirsutum and G. raimondii. The ‘GosTE’ involved in the capture and proliferation of MIR482/2118d and its derivatives belongs to the PIF/Harbinger superfamily, generating a 3‐bp target site duplication upon insertion at new locations. All orthologous MIR482/2118 loci in the two diploids were retained in the two tetraploids, but mutation(s) in miR482/2118 were observed across all four species as well as in different cultivars of both G. barbadense and G. hirsutum, suggesting a dynamic co‐evolution of miR482/2118 and its NBS‐LRR targets. Our results provide fresh insights into the mechanisms contributing to MIRNA proliferation and enrich our knowledge on TEs.