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Protein phosphorylation is a major molecular switch involved in the regulation of stomatal opening and closure. Previous research defined interaction between MAP kinase 12 and Raf‐like kinase HT1 as a required step for stomatal movements caused by changes in CO₂concentration. However, whether MPK12 kinase activity is required for regulation of CO₂‐induced stomatal responses warrants in‐depth investigation.

We apply genetic, biochemical, and structural modeling approaches to examining the noncatalytic role of MPK12 in guard cell CO₂signaling that relies on allosteric inhibition of HT1.

We show that CO₂/HCO₃⁻‐enhanced MPK12 interaction with HT1 is independent of its kinase activity. By analyzing gas exchange of plant lines expressing various kinase‐dead and constitutively active versions of MPK12 in a plant line whereMPK12is deleted, we confirmed that CO₂‐dependent stomatal responses rely on MPK12's ability to bind to HT1, but not its kinase activity. We also demonstrate that purified MPK12 and HT1 proteins form a heterodimer in the presence of CO₂/HCO₃⁻and present structural modeling that explains the MPK12:HT1 interaction interface.

These data add to the model that MPK12 kinase‐activity‐independent interaction with HT1 functions as a molecular switch by which guard cells sense changes in atmospheric CO₂concentration.

 

Little is known about long‐distance mesophyll‐driven signals that regulate stomatal conductance. Soluble and/or vapor‐phase molecules have been proposed. In this study, the involvement of the gaseous signal ethylene in the modulation of stomatal conductance inArabidopsis thalianaby CO₂/abscisic acid (ABA) was examined.

We present a diffusion model which indicates that gaseous signaling molecule/s with a shorter/direct diffusion pathway to guard cells are more probable for rapid mesophyll‐dependent stomatal conductance changes. We, therefore, analyzed different Arabidopsis ethylene‐signaling and biosynthesis mutants for their ethylene production and kinetics of stomatal responses to ABA/[CO₂]‐shifts.

According to our research, higher [CO₂] causes Arabidopsis rosettes to produce more ethylene. An ACC‐synthase octuple mutant with reduced ethylene biosynthesis exhibits dysfunctional CO₂‐induced stomatal movements. Ethylene‐insensitive receptor (gain‐of‐function),etr1‐1andetr2‐1, and signaling,ein2‐5andein2‐1, mutants showed intact stomatal responses to [CO₂]‐shifts, whereas loss‐of‐function ethylene receptor mutants, includingetr2‐3;ein4‐4;ers2‐3,etr1‐6;etr2‐3andetr1‐6, showed markedly accelerated stomatal responses to [CO₂]‐shifts. Further investigation revealed a significantly impaired stomatal closure to ABA in the ACC‐synthase octuple mutant and accelerated stomatal responses in theetr1‐6;etr2‐3, andetr1‐6, but not in theetr2‐3;ein4‐4;ers2‐3mutants.

These findings suggest essential functions of ethylene biosynthesis and signaling components in tuning/accelerating stomatal conductance responses to CO₂and ABA.

 

We present unresolved questions in plant abiotic stress biology as posed by 15 research groups with expertise spanning eco-physiology to cell and molecular biology. Common themes of these questions include the need to better understand how plants detect water availability, temperature, salinity, and rising carbon dioxide (CO2) levels; how environmental signals interface with endogenous signaling and development (e.g. circadian clock and flowering time); and how this integrated signaling controls downstream responses (e.g. stomatal regulation, proline metabolism, and growth versus defense balance). The plasma membrane comes up frequently as a site of key signaling and transport events (e.g. mechanosensing and lipid-derived signaling, aquaporins). Adaptation to water extremes and rising CO2 affects hydraulic architecture and transpiration, as well as root and shoot growth and morphology, in ways not fully understood. Environmental adaptation involves tradeoffs that limit ecological distribution and crop resilience in the face of changing and increasingly unpredictable environments. Exploration of plant diversity within and among species can help us know which of these tradeoffs represent fundamental limits and which ones can be circumvented by bringing new trait combinations together. Better defining what constitutes beneficial stress resistance in different contexts and making connections between genes and phenotypes, and between laboratory and field observations, are overarching challenges.

 

Jasmonic acid (JA) and salicylic acid (SA) regulate stomatal closure, preventing pathogen invasion into plants. However, to what extent abscisic acid (ABA), SA and JA interact, and what the roles of SA and JA are in stomatal responses to environmental cues, remains unclear. Here, by using intact plant gas‐exchange measurements in JA and SA single and double mutants, we show that stomatal responsiveness to CO₂, light intensity, ABA, high vapor pressure deficit and ozone either did not or, for some stimuli only, very slightly depended upon JA and SA biosynthesis and signaling mutants, includingdde2, sid2, coi1,jai1,myc2andnpr1alleles. Although the stomata in the mutants studied clearly responded to ABA, CO₂, light and ozone, ABA‐triggered stomatal closure innpr1‐1was slightly accelerated compared with the wild type. Stomatal reopening after ozone pulses was quicker in thecoi1‐16mutant than in the wild type. In intact Arabidopsis plants, spraying with methyl‐JA led to only a modest reduction in stomatal conductance 80 min after treatment, whereas ABA and CO₂induced pronounced stomatal closure within minutes. We could not document a reduction of stomatal conductance after spraying with SA. Coronatine‐induced stomatal opening was initiated slowly after 1.5–2.0 h, and reached a maximum by 3 h after spraying intact plants. Our results suggest that ABA, CO₂and light are major regulators of rapid guard cell signaling, whereas JA and SA could play only minor roles in the whole‐plant stomatal response to environmental cues in Arabidopsis andSolanum lycopersicum(tomato).

 

Abiotic stresses, including drought and salinity, trigger a complex osmotic-stress and abscisic acid (ABA) signal transduction network. The core ABA signalling components are snf1-related protein kinase2s (SnRK2s), which are activated by ABA-triggered inhibition of type-2C protein-phosphatases (PP2Cs). SnRK2 kinases are also activated by a rapid, largely unknown, ABA-independent osmotic-stress signalling pathway. Here, through a combination of a redundancy-circumventing genetic screen and biochemical analyses, we have identified functionally-redundant MAPKK-kinases (M3Ks) that are necessary for activation of SnRK2 kinases. These M3Ks phosphorylate a specific SnRK2/OST1 site, which is indispensable for ABA-induced reactivation of PP2C-dephosphorylated SnRK2 kinases. ABA-triggered SnRK2 activation, transcription factor phosphorylation and SLAC1 activation require these M3Ks in vitro and in plants. M3K triple knock-out plants show reduced ABA sensitivity and strongly impaired rapid osmotic-stress-induced SnRK2 activation. These findings demonstrate that this M3K clade is required for ABA- and osmotic-stress-activation of SnRK2 kinases, enabling robust ABA and osmotic stress signal transduction.

 

Low concentrations of CO₂cause stomatal opening, whereas [CO₂] elevation leads to stomatal closure. Classical studies have suggested a role for Ca²⁺and protein phosphorylation in CO₂‐induced stomatal closing. Calcium‐dependent protein kinases (CPKs) and calcineurin‐B‐like proteins (CBLs) can sense and translate cytosolic elevation of the second messenger Ca²⁺into specific phosphorylation events. However, Ca²⁺‐binding proteins that function in the stomatal CO₂response remain unknown.

Time‐resolved stomatal conductance measurements using intact plants, and guard cell patch‐clamp experiments were performed.

We isolatedcpkquintuple mutants and analyzed stomatal movements in response to CO₂, light and abscisic acid (ABA). Interestingly, we found thatcpk3/5/6/11/23quintuple mutant plants, but not other analyzedcpkquadruple/quintuple mutants, were defective in high CO₂‐induced stomatal closure and, unexpectedly, also in low CO₂‐induced stomatal opening. Furthermore, K⁺‐uptake‐channel activities were reduced incpk3/5/6/11/23quintuple mutants, in correlation with the stomatal opening phenotype. However, light‐mediated stomatal opening remained unaffected, and ABA responses showed slowing in some experiments. By contrast, CO₂‐regulated stomatal movement kinetics were not clearly affected in plasma membrane‐targetedcbl1/4/5/8/9quintuple mutant plants.

Our findings describe combinatorialcpkmutants that function in CO₂control of stomatal movements and support the results of classical studies showing a role for Ca²⁺in this response.

 

Urban community gardens have increased in prevalence as a means to generate fresh fruits and vegetables, including in areas lacking access to healthy food options. However, urban soils may have high levels of toxic heavy metals, including lead and cadmium and the metalloid arsenic, which can lead to severe health risks. In this study, fruit and vegetable samples grown at an urban community garden in southeastern San Diego, the Ocean View Growing Grounds, were sampled repeatedly over a four‐year time period in order to measure potential contamination of toxic heavy metals and metalloids and to develop solutions for this problem. Metal nutrient, heavy metal, and metalloid concentrations were monitored in the leaf and fruit tissues of fruit trees over the sampling period. Several of the fruit trees showed uptake of lead in the leaf samples, with Black Mission fig measuring 0.843–1.531 mg/kg dry weight and Mexican Lime measuring 1.103–1.522 mg/kg dry weight over the sampling period. Vegetables that were grown directly in the ground at this community garden and surrounding areas showed arsenic, 0.80 + 0.073 mg/kg dry weight for Swiss chard, and lead, 0.84 ± 0.404 mg/kg dry weight for strawberries, in their edible tissues. The subsequent introduction of raised beds with uncontaminated soil is described, which eliminated any detectable heavy metal or metalloid contamination in these crops during the monitoring period. Recommendations for facilitating the monitoring of edible tissues and for reducing risk are discussed, including introduction of raised beds and collaborations with local universities and research groups.

 

Plant stomata sense CO2 via reversible interaction of the Raf-like HT1 protein kinase with non-activity requiring MAP kinase. 

In plants, vegetative and reproductive development are associated with agronomically important traits that contribute to grain yield and biomass. Zinc finger homeodomain (ZF-HD) transcription factors (TFs) constitute a relatively small gene family that has been studied in several model plants, including Arabidopsis thaliana L. and Oryza sativa L. The ZF-HD family members play important roles in plant growth and development, but their contribution to the regulation of plant architecture remains largely unknown due to their functional redundancy. To understand the gene regulatory network controlled by ZF-HD TFs, we analyzed multiple loss-of-function mutants of ZF-HD TFs in Arabidopsis that exhibited morphological abnormalities in branching and flowering architecture. We found that ZF-HD TFs, especially HB34, negatively regulate the expression of miR157 and positively regulate SQUAMOSA PROMOTER BINDING–LIKE 10 (SPL10), a target of miR157. Genome-wide chromatin immunoprecipitation sequencing (ChIP-Seq) analysis revealed that miR157D and SPL10 are direct targets of HB34, creating a feed-forward loop that constitutes a robust miRNA regulatory module. Network motif analysis contains overrepresented coherent type IV feedforward motifs in the amiR zf-HD and hbq mutant background. This finding indicates that miRNA-mediated ZF-HD feedforward modules modify branching and inflorescence architecture in Arabidopsis. Taken together, these findings reveal a guiding role of ZF-HD TFs in the regulatory network module and demonstrate its role in plant architecture in Arabidopsis.