ZEITLUPE Promotes ABA-Induced Stomatal Closure in Arabidopsis and Populus.

Plants balance water availability with gas exchange and photosynthesis by controlling stomatal aperture. This control is regulated in part by the circadian clock, but it remains unclear how signalling pathways of daily rhythms are integrated into stress responses. The serine/threonine protein kinase OPEN STOMATA 1 (OST1) contributes to the regulation of stomatal closure via activation of S-type anion channels. OST1 also mediates gene regulation in response to ABA/drought stress. We show that ZEITLUPE (ZTL), a blue light photoreceptor and clock component, also regulates ABA-induced stomatal closure in Arabidopsis thaliana, establishing a link between clock and ABA-signalling pathways. ZTL sustains expression of OST1 and ABA-signalling genes. Stomatal closure in response to ABA is reduced in ztl mutants, which maintain wider stomatal apertures and show higher rates of gas exchange and water loss than wild-type plants. Detached rosette leaf assays revealed a stronger water loss phenotype in ztl-3, ost1-3 double mutants, indicating that ZTL and OST1 contributed synergistically to the control of stomatal aperture. Experimental studies of Populus sp., revealed that ZTL regulated the circadian clock and stomata, indicating ZTL function was similar in these trees and Arabidopsis. PSEUDO-RESPONSE REGULATOR 5 (PRR5), a known target of ZTL, affects ABA-induced responses, including stomatal regulation. Like ZTL, PRR5 interacted physically with OST1 and contributed to the integration of ABA responses with circadian clock signalling. This suggests a novel mechanism whereby the PRR proteins-which are expressed from dawn to dusk-interact with OST1 to mediate ABA-dependent plant responses to reduce water loss in time of stress.

CO2 Sensing and CO2 Regulation of Stomatal Conductance: Advances and Open Questions.

Guard cells form epidermal stomatal gas-exchange valves in plants and regulate the aperture of stomatal pores in response to changes in the carbon dioxide (CO2) concentration ([CO2]) in leaves. Moreover, the development of stomata is repressed by elevated CO2 in diverse plant species. Evidence suggests that plants can sense [CO2] changes via guard cells and via mesophyll tissues in mediating stomatal movements. We review new discoveries and open questions on mechanisms mediating CO2-regulated stomatal movements and CO2 modulation of stomatal development, which together function in the CO2 regulation of stomatal conductance and gas exchange in plants. Research in this area is timely in light of the necessity of selecting and developing crop cultivars that perform better in a shifting climate.

The HT1 protein kinase is essential for red light-induced stomatal opening and genetically interacts with OST1 in red light and CO2 -induced stomatal movement responses.

The question of whether red light-induced stomatal opening is mediated by a photosynthesis-derived reduction in intercellular [CO2 ] (Ci ) remains controversial and genetic analyses are needed. The Arabidopsis thaliana protein kinase HIGH TEMPERATURE 1 (HT1) is a negative regulator of [CO2 ]-induced stomatal closing and ht1-2 mutant plants do not show stomatal opening to low [CO2 ]. The protein kinase mutant ost1-3 exhibits slowed stomatal responses to CO2 . The functions of HT1 and OPEN STOMATA 1 (OST1) to changes in red, blue light or [CO2 ] were analyzed. For comparison we assayed recessive ca1ca4 carbonic anhydrase double mutant plants, based on their slowed stomatal response to CO2 . Here, we report a strong impairment in ht1 in red light-induced stomatal opening whereas blue light was able to induce stomatal opening. The effects on photosynthetic performance in ht1 were restored when stomatal limitation of CO2 uptake, by control of [Ci ], was eliminated. HT1 was found to interact genetically with OST1 both during red light- and low [CO2 ]-induced stomatal opening. Analyses of ca1ca4 plants suggest that more than a low [Ci ]-dependent pathway may function in red light-induced stomatal opening. These results demonstrate that HT1 is essential for red light-induced stomatal opening and interacts genetically with OST1 during stomatal responses to red light and altered [CO2 ].

Guard cell photosynthesis is critical for stomatal turgor production, yet does not directly mediate CO2 - and ABA-induced stomatal closing.

Stomata mediate gas exchange between the inter-cellular spaces of leaves and the atmosphere. CO2 levels in leaves (Ci) are determined by respiration, photosynthesis, stomatal conductance and atmospheric [CO2 ]. [CO2 ] in leaves mediates stomatal movements. The role of guard cell photosynthesis in stomatal conductance responses is a matter of debate, and genetic approaches are needed. We have generated transgenic Arabidopsis plants that are chlorophyll-deficient in guard cells only, expressing a constitutively active chlorophyllase in a guard cell specific enhancer trap line. Our data show that more than 90% of guard cells were chlorophyll-deficient. Interestingly, approximately 45% of stomata had an unusual, previously not-described, morphology of thin-shaped chlorophyll-less stomata. Nevertheless, stomatal size, stomatal index, plant morphology, and whole-leaf photosynthetic parameters (PSII, qP, qN, FV '/FM' ) were comparable with wild-type plants. Time-resolved intact leaf gas-exchange analyses showed a reduction in stomatal conductance and CO2 -assimilation rates of the transgenic plants. Normalization of CO2 responses showed that stomata of transgenic plants respond to [CO2 ] shifts. Detailed stomatal aperture measurements of normal kidney-shaped stomata, which lack chlorophyll, showed stomatal closing responses to [CO2 ] elevation and abscisic acid (ABA), while thin-shaped stomata were continuously closed. Our present findings show that stomatal movement responses to [CO2 ] and ABA are functional in guard cells that lack chlorophyll. These data suggest that guard cell CO2 and ABA signal transduction are not directly modulated by guard cell photosynthesis/electron transport. Moreover, the finding that chlorophyll-less stomata cause a 'deflated' thin-shaped phenotype, suggests that photosynthesis in guard cells is critical for energization and guard cell turgor production.

Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield.

BACKGROUND: Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals. RESULTS: We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment. CONCLUSIONS: Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass.

Carbonic anhydrases are upstream regulators of CO2-controlled stomatal movements in guard cells.

The continuing rise in atmospheric CO2 causes stomatal pores in leaves to close and thus globally affects CO2 influx into plants, water use efficiency and leaf heat stress. However, the CO2-binding proteins that control this response remain unknown. Moreover, which cell type responds to CO2, mesophyll or guard cells, and whether photosynthesis mediates this response are matters of debate. We demonstrate that Arabidopsis thaliana double-mutant plants in the beta-carbonic anhydrases betaCA1 and betaCA4 show impaired CO2-regulation of stomatal movements and increased stomatal density, but retain functional abscisic-acid and blue-light responses. betaCA-mediated CO2-triggered stomatal movements are not, in first-order, linked to whole leaf photosynthesis and can function in guard cells. Furthermore, guard cell betaca-overexpressing plants exhibit instantaneous enhanced water use efficiency. Guard cell expression of mammalian alphaCAII complements the reduced sensitivity of ca1 ca4 plants, showing that carbonic anhydrase-mediated catalysis is an important mechanism for betaCA-mediated CO2-induced stomatal closure and patch clamp analyses indicate that CO2/HCO3- transfers the signal to anion channel regulation. These findings, together with ht1-2 (ref. 9) epistasis analysis demonstrate that carbonic anhydrases function early in the CO2 signalling pathway, which controls gas-exchange between plants and the atmosphere.