Arabidopsis VILLIN5 bundles actin filaments using a novel mechanism.

Being a bona fide actin bundler, Arabidopsis villin5 (VLN5) plays a crucial role in regulating actin stability and organization within pollen tubes. Despite its significance, the precise mechanism through which VLN5 bundles actin filaments has remained elusive. Through meticulous deletion analysis, we have unveiled that the link between gelsolin repeat 6 (G6) and the headpiece domain (VHP), rather than VHP itself, is indispensable for VLN5-mediated actin bundling. Further refinement of this region has pinpointed a critical sequence spanning from Val763 to Ser823, essential for VLN5's actin-bundling activity. Notably, the absence of Val763-Ser823 in VLN5 results in decreased filamentous decoration within pollen tubes and a diminished ability to rescue actin bundling defects in vln2vln5 mutant pollen tubes compared to intact VLN5. Moreover, our findings highlight that the Val763-Ser823 sequence harbors a binding site for F-actin, suggesting that this linker-based F-actin binding site, in conjunction with the F-actin binding site localized in G1-G6, enables a single VLN5 to concurrently bind to two adjacent actin filaments. Therefore, our study unveils a novel mechanism by which VLN5 bundles actin filaments.

Arabidopsis thaliana MYC2 and MYC3 Are Involved in Ethylene-Regulated Hypocotyl Growth as Negative Regulators.

The ethylene-regulated hypocotyl elongation of Arabidopsis thaliana involves many transcription factors. The specific role of MYC transcription factors in ethylene signal transduction is not completely understood. The results here revealed that two MYCs, MYC2 and MYC3, act as negative regulators in ethylene-suppressed hypocotyl elongation. Etiolated seedlings of the loss-of-function mutant of MYC2 or MYC3 were significantly longer than wild-type seedlings. Single- or double-null mutants of MYC2 and MYC3 displayed remarkably enhanced response to ACC(1-aminocyclopropane-1-carboxylate), the ethylene precursor, compared to wild-type seedlings. MYC2 and MYC3 directly bind to the promoter zone of ERF1, strongly suppressing its expression. Additionally, EIN3, a key component in ethylene signaling, interacts with MYC2 or MYC3 and significantly suppresses their binding to ERF1's promoter. MYC2 and MYC3 play crucial roles in the ethylene-regulated expression of functional genes. The results revealed the novel role and functional mechanism of these transcription factors in ethylene signal transduction. The findings provide valuable information for deepening our understanding of their role in regulating plant growth and responding to stress.

Gibberellin Positively Regulates Tomato Resistance to Tomato Yellow Leaf Curl Virus (TYLCV).

Tomato yellow leaf curl virus (TYLCV) is a prominent viral pathogen that adversely affects tomato plants. Effective strategies for mitigating the impact of TYLCV include isolating tomato plants from the whitefly, which is the vector of the virus, and utilizing transgenic lines that are resistant to the virus. In our preliminary investigations, we observed that the use of growth retardants increased the rate of TYLCV infection and intensified the damage to the tomato plants, suggesting a potential involvement of gibberellic acid (GA) in the conferring of resistance to TYLCV. In this study, we employed an infectious clone of TYLCV to inoculate tomato plants, which resulted in leaf curling and growth inhibition. Remarkably, this inoculation also led to the accumulation of GA3 and several other phytohormones. Subsequent treatment with GA3 effectively alleviated the TYLCV-induced leaf curling and growth inhibition, reduced TYLCV abundance in the leaves, enhanced the activity of antioxidant enzymes, and lowered the reactive oxygen species (ROS) levels in the leaves. Conversely, the treatment with PP333 exacerbated TYLCV-induced leaf curling and growth suppression, increased TYLCV abundance, decreased antioxidant enzyme activity, and elevated ROS levels in the leaves. The analysis of the gene expression profiles revealed that GA3 up-regulated the genes associated with disease resistance, such as WRKYs, NACs, MYBs, Cyt P450s, and ERFs, while it down-regulated the DELLA protein, a key agent in GA signaling. In contrast, PP333 induced gene expression changes that were the opposite of those caused by the GA3 treatment. These findings suggest that GA plays an essential role in the tomato's defense response against TYLCV and acts as a positive regulator of ROS scavenging and the expression of resistance-related genes.

ATANN3 Is Involved in Extracellular ATP-Regulated Auxin Distribution in Arabidopsis thaliana Seedlings.

Extracellular ATP (eATP) plays multiple roles in plant growth and development, and stress responses. It has been revealed that eATP suppresses growth and alters the growth orientation of the root and hypocotyl of Arabidopsis thaliana by affecting auxin transport and localization in these organs. However, the mechanism of the eATP-stimulated auxin distribution remains elusive. Annexins are involved in multiple aspects of plant cellular metabolism, while their role in response to apoplastic signals remains unclear. Here, by using the loss-of-function mutations, we investigated the role of AtANN3 in the eATP-regulated root and hypocotyl growth. Firstly, the inhibitory effects of eATP on root and hypocotyl elongation were weakened or impaired in the AtANN3 null mutants (atann3-1 and atann3-2). Meanwhile, the distribution of DR5-GUS and DR5-GFP indicated that the eATP-induced asymmetric distribution of auxin in the root tips or hypocotyl cells occurred in wild-type control plants, while in atann3-1 mutant seedlings, it was not observed. Further, the eATP-induced asymmetric distribution of PIN2-GFP in root-tip cells or that of PIN3-GFP in hypocotyl cells was reduced in atann3-1 seedlings. Finally, the eATP-induced asymmetric distribution of cytoplasmic vesicles in root-tip cells was impaired in atann3-1 seedlings. Based on these results, we suggest that AtANN3 may be involved in eATP-regulated seedling growth by regulating the distribution of auxin and auxin transporters in vegetative organs.

Changing surface wax compositions and related gene expression in three cultivars of Chinese pear fruits during cold storage.

The surface wax of fruit has a significant effect on abiotic stress and fruit quality. In this study, the composition of the waxes found on fruit surfaces and the related gene expression of three different pear cultivars (Xuehua, Yali, and Yuluxiang) were investigated during cold storage. The results showed that 35 wax compositions were found on the surfaces of the three pear cultivars, mainly including C29 alkane, three fatty acids, two esters, three aldehydes, three fatty alcohols, and three triterpenoids. The largest amount of C29 alkane, three fatty acids and two esters were found in Yuluxiang (YLX) on day 90, while aldehydes with carbons of C30 and C32 were the highest in Yali (YL). Xuehua (XH) showed the largest amount of C22 fatty alcohol on day 180 compared to YLX and YL. Larger amounts of triterpenoids were found in XH and YL when compared to YLX. The expression levels of fifteen wax related genes (LACS1, KCS2, KCS6, FDH, KCS20, GL8, CER10, CER60, LTPG1, LTP4, ABCG12, CER1L, CAC3, CAC3L, and DGAT1L) reached their peak at day 45 in YLX, compared to XH and YL, their expression levels in YLX were higher to different degrees. These results suggest that the different expression patterns of wax-related genes may be closely related to the difference in wax compositions of the surface wax of three pear cultivars.

RNA-Seq Analysis Identifies Transcription Factors Involved in Anthocyanin Biosynthesis of 'Red Zaosu' Pear Peel and Functional Study of PpPIF8.

Red-skinned pears are favored by people for their attractive appearance and abundance of anthocyanins. However, the molecular basis of anthocyanin biosynthesis in red pears remains elusive. Here, a comprehensive transcriptome analysis was conducted to explore the potential regulatory mechanism of anthocyanin biosynthesis in 'Red Zaosu' pear (Pyrus pyrifolia × Pyrus communis). Gene co-expression analysis and transcription factor mining identified 263 transcription factors, which accounted for 6.59% of the total number of transcription factors in the pear genome in two gene modules that are highly correlated with anthocyanin biosynthesis. Clustering, gene network modeling with STRING-DB, and local motif enrichment analysis (CentriMo) analysis suggested that PpPIF8 may play a role in anthocyanin biosynthesis. Furthermore, eight PIFs were identified in the pear genome, of which only PpPIF8 was rapidly induced by light. Functional studies showed that PpPIF8 localizes in the nucleus and is preferentially expressed in the tissue of higher levels of anthocyanin. The overexpression of PpPIF8 in pear peel and pear calli promotes anthocyanin biosynthesis and upregulates the expression of anthocyanin biosynthesis genes. Yeast-one hybrid and transgenic analyses indicated that PpPIF8 binds to the PpCHS promoter to induce PpCHS expression. The positive effect of PpPIF8 on anthocyanin biosynthesis is different from previously identified negative regulators of PyPIF5 and MdPIF7 in pear and apple. Taken together, our data not only provide a comprehensive view of transcription events during the coloration of pear peel, but also resolved the regulatory role of PpPIF8 in the anthocyanin biosynthesis pathway.

AtANN1 and AtANN2 are involved in phototropism of etiolated hypocotyls of Arabidopsis by regulating auxin distribution.

Phototropism is an essential response in some plant organs and features several signalling molecules involved in either photo-sensing or post-sensing responses. Annexins are involved in regulating plant growth and its responses to various stimuli. Here, we provide novel data showing that two members of the Annexin family in Arabidopsis thaliana, AtANN1 and AtANN2, may be involved in the phototropism of etiolated hypocotyls. In wild type, unilateral blue light (BL) induced a strong phototropic response, while red light (RL) only induced a weak response. The responses of single- or double-null mutants of the two annexins, including atann1, atann2 and atann1/atann2, were significantly weaker than those observed in wild type, indicating the involvement of AtANN1 and AtANN2 in BL-induced phototropism. Unilateral BL induced asymmetric distribution of DR5-GFP and PIN3-GFP fluorescence in hypocotyls; notably, fluorescent intensity on the shaded side was markedly stronger than that on the illuminated side. In etiolated atann1, atann2 or atann1/atann2 hypocotyls, unilateral BL-induced asymmetric distributions of DR5-GFP and PIN3-GFP were weakened or impaired. Herein, we suggest that during hypocotyls phototropic response, AtANN1 and AtANN2 may be involved in BL-stimulated signalling by regulating PIN3-charged auxin transport.

Kinase activity is required for the receptor kinase DROOPY LEAF1 to control leaf droopiness.

Plants have evolved many leucine-rich repeat receptor-like kinases (LRR-RLKs) that control all aspects of plant life in a kinase-dependent or -independent manner. DROOPY LEAF1 (DPY1), which is a subfamily II LRR-RLK authentic kinase, controls leaf droopiness by negatively regulating early brassinosteroid (BR) signaling in foxtail millet. In this study, we proved that overexpressing kinase-inactive DPY1 does not rescue the droopy leaf phenotype of dpy1 plants because the mutated DPY1 cannot repress BR signaling, suggesting that kinase activity is required for DPY1 to control BR signaling. Moreover, seven DPY1 sites potentially transphosphorylated by SiBAK1 were identified as crucial for DPY1 activation. These findings highlight the importance of kinase activity for the functionality of DPY1.

P2K1 Receptor, Heterotrimeric Gα Protein and CNGC2/4 Are Involved in Extracellular ATP-Promoted Ion Influx in the Pollen of Arabidopsis thaliana.

As an apoplastic signal, extracellular ATP (eATP) is involved in plant growth and development. eATP promotes tobacco pollen germination (PG) and pollen tube growth (PTG) by stimulating Ca2+ or K+ absorption. Nevertheless, the mechanisms underlying eATP-stimulated ion uptake and their role in PG and PTG are still unclear. Here, ATP addition was found to modulate PG and PTG in 34 plant species and showed a promoting effect in most of these species. Furthermore, by using Arabidopsis thaliana as a model, the role of several signaling components involved in eATP-promoted ion (Ca2+, K+) uptake, PG, and PTG were investigated. ATP stimulated while apyrase inhibited PG and PTG. Patch-clamping results showed that ATP promoted K+ and Ca2+ influx into pollen protoplasts. In loss-of-function mutants of P2K1 (dorn1-1 and dorn1-3), heterotrimeric G protein α subunit (gpa1-1, gpa1-2), or cyclic nucleotide gated ion channel (cngc2, cngc4), eATP-stimulated PG, PTG, and ion influx were all impaired. Our results suggest that these signaling components may be involved in eATP-promoted PG and PTG by regulating Ca2+ or K+ influx in Arabidopsis pollen grains.

RRFT1 (Redox Responsive Transcription Factor 1) is involved in extracellular ATP-regulated gene expression in Arabidopsis thaliana seedlings.

As an apoplast signal molecule, extracellular ATP (eATP) is involved in the growth regulation of Arabidopsis thaliana seedlings. Recently, RRFT1 was revealed to be involved in eATP- regulated seedling growth. To further verify the role of RRTF1 in seedlings' eATP response, expression of 20 eATP-responsive genes in wild type (Col-0) and RRTF1 null mutant (rrtf1-1) seedlings were investigated by using realtime quantitative PCR. After 0.5 mM ATP stimulation, the response of these genes' expression in rrtf1-1 seedlings was significantly different from that in Col-0 seedlings. Proteins which are encoded by these genes include transcription factors, plasma membrane receptors like kinases, ion influx/efflux transporters and hormone signaling components. The results indicated that RRTF1 may be involved in eATP regulated physiological responses via regulating the expression of some functional genes.

Redox-Responsive Transcription Factor 1 (RRFT1) Is Involved in Extracellular ATP-Regulated Arabidopsis thaliana Seedling Growth.

Extracellular adenosine triphosphate (eATP) is an apoplastic signaling molecule that plays an essential role in the growth and development of plants. Arabidopsis seedlings have been reported to respond to eATP; however, the downstream signaling components are still not well understood. In this study, we report that an ethylene-responsive factor, Redox-Responsive Transcription Factor 1 (RRTF1), is involved in eATP-regulated Arabidopsis thaliana seedling growth. Exogenous adenosine triphosphate inhibited green seedling root growth and induced hypocotyl bending of etiolated seedlings. RRTF1 loss-of-function mutant (rrtf1) seedlings showed decreased responses to eATP, while its complementation or overexpression led to recovered or increased eATP responsiveness. RRTF1 was expressed rapidly after eATP stimulation and then migrated into the nuclei of root tip cells. eATP-induced auxin accumulation in root tip or hypocotyl cells was impaired in rrtf1. Chromatin immunoprecipitation and high-throughput sequencing results indicated that eATP induced some genes related to cell growth and development in wild type but not in rrtf1 cells. These results suggest that RRTF1 may be involved in eATP signaling by regulating functional gene expression and cell metabolism in Arabidopsis seedlings.

Arabidopsis cyclic nucleotide-gated channel 6 is negatively modulated by multiple calmodulin isoforms during heat shock.

An increased concentration of cytosolic Ca2+ is an early response of plant cells to heat shock. Arabidopsis cyclic nucleotide-gated ion channel 6 (CNGC6) mediates heat-induced Ca2+ influx and is activated by cAMP. However, it remains unclear how the Ca2+ conductivity of CNGC6 is negatively regulated under the elevated cytosolic Ca2+ concentration. In this study, Arabidopsis calmodulin isoforms CaM1/4, CaM2/3/5, CaM6, and CaM7 were found to bind to CNGC6 to varying degrees, and this binding was dependent on the presence of Ca2+ and IQ6, an atypical isoleucine-glutamine motif in CNGC6. Knockout of CaM2, CaM3, CaM5, and CaM7 genes led to a marked increase in plasma membrane inward Ca2+ current under heat shock conditions; however, knockout of CaM1, CaM4, and CaM6 genes had no significant effect on plasma membrane Ca2+ current. Moreover, the deletion of IQ6 from CNGC6 led to a marked increase in plasma membrane Ca2+ current under heat shock conditions. Taken together, the data suggest that CNGC6-mediated Ca2+ influx is likely to be negatively regulated by CaM2/3/5 and CaM7 isoforms under heat shock conditions, and that IQ6 plays an important role in CaM binding and the feedback regulation of the channel.

Extracellular ATP promoted pollen germination and tube growth of Nicotiana tabacum through promoting K+ and Ca2+ absorption.

Extracellular ATP (eATP) plays an essential role in plant growth, development, and stress tolerance. Here, we report that eATP participated in Nicotiana tabacum pollen germination (PG) and pollen tube growth (PTG) by regulating K+ and Ca2+ influx. Exogenous ATP or ADP effectively promoted PG and PTG in a dose-dependent manner; weakly hydrolysable ATP analog (ATPγS) showed a similar effect. AMP, adenosine, adenine, and phosphate did not affect PG or PTG. Within a certain range, higher concentrations of K+ or Ca2+ in the medium increased the effect of ATP in promoting PG and PTG. However, in mediums containing K+ or Ca2+ concentrations above this range, the effect of ATP was reversed, resulting in PG and PTG inhibition. Ca2+ chelators (EGTA), Ca2+ channel blockers, and K+ channel blockers suppressed ATP-promoted PG and PTG. Results from a patch clamp showed that ATP activated a K+ and Ca2+ influx in pollen protoplasts. These results suggest that, as an apoplastic signal, eATP may be involved in PG and PTG via regulating Ca2+ and K+ absorption.

Heterotrimeric G Protein-Regulated Ca2+ Influx and PIN2 Asymmetric Distribution Are Involved in Arabidopsis thaliana Roots' Avoidance Response to Extracellular ATP.

Extracellular ATP (eATP) has been reported to be involved in plant growth as a primary messenger in the apoplast. Here, roots of Arabidopsis thaliana seedlings growing in jointed medium bent upon contact with ATP-containing medium to keep away from eATP, showing a marked avoidance response. Roots responded similarly to ADP and bz-ATP but did not respond to AMP and GTP. The eATP avoidance response was reduced in loss-of-function mutants of heterotrimeric G protein α subunit (Gα) (gpa1-1 and gpa1-2) and enhanced in Gα-over-expression (OE) lines (wGα and cGα). Ethylenebis(oxyethylenenitrilo) tetraacetic acid (EGTA) and Gd3+ remarkably suppressed eATP-induced root bending. ATP-stimulated Ca2+ influx was impaired in Gα null mutants and increased in its OE lines. DR5-GFP and PIN2 were asymmetrically distributed in ATP-stimulated root tips, this effect was strongly suppressed by EGTA and diminished in Gα null mutants. In addition, some eATP-induced genes' expression was also impaired in Gα null mutants. Based on these results, we propose that heterotrimeric Gα-regulated Ca2+ influx and PIN2 distribution may be key signaling events in eATP sensing and avoidance response in Arabidopsis thaliana roots.

Classification and identification of Rhodobryum roseum Limpr. and its adulterants based on fourier-transform infrared spectroscopy (FTIR) and chemometrics.

Fourier-transform infrared spectroscopy (FTIR) with the attenuated total reflectance technique was used to identify Rhodobryum roseum from its four adulterants. The FTIR spectra of six samples in the range from 4000 cm-1 to 600 cm-1 were obtained. The second-derivative transformation test was used to identify the small and nearby absorption peaks. A cluster analysis was performed to classify the spectra in a dendrogram based on the spectral similarity. Principal component analysis (PCA) was used to classify the species of six moss samples. A cluster analysis with PCA was used to identify different genera. However, some species of the same genus exhibited highly similar chemical components and FTIR spectra. Fourier self-deconvolution and discrete wavelet transform (DWT) were used to enhance the differences among the species with similar chemical components and FTIR spectra. Three scales were selected as the feature-extracting space in the DWT domain. The results show that FTIR spectroscopy with chemometrics is suitable for identifying Rhodobryum roseum and its adulterants.

The Arabidopsis RCC1 Family Protein TCF1 Regulates Freezing Tolerance and Cold Acclimation through Modulating Lignin Biosynthesis.

Cell water permeability and cell wall properties are critical to survival of plant cells during freezing, however the underlying molecular mechanisms remain elusive. Here, we report that a specifically cold-induced nuclear protein, Tolerant to Chilling and Freezing 1 (TCF1), interacts with histones H3 and H4 and associates with chromatin containing a target gene, blue-copper-binding protein (BCB), encoding a glycosylphosphatidylinositol-anchored protein that regulates lignin biosynthesis. Loss of TCF1 function leads to reduced BCB transcription through affecting H3K4me2 and H3K27me3 levels within the BCB gene, resulting in reduced lignin content and enhanced freezing tolerance. Furthermore, plants with knocked-down BCB expression (amiRNA-BCB) under cold acclimation had reduced lignin accumulation and increased freezing tolerance. The pal1pal2 double mutant (lignin content reduced by 30% compared with WT) also showed the freezing tolerant phenotype, and TCF1 and BCB act upstream of PALs to regulate lignin content. In addition, TCF1 acts independently of the CBF (C-repeat binding factor) pathway. Our findings delineate a novel molecular pathway linking the TCF1-mediated cold-specific transcriptional program to lignin biosynthesis, thus achieving cell wall remodeling with increased freezing tolerance.

A calcium-binding protein, rice annexin OsANN1, enhances heat stress tolerance by modulating the production of H2O2.

OsANN1 is a member of the annexin protein family in rice. The function of this protein and the mechanisms of its involvement in stress responses and stress tolerance are largely unknown. Here it is reported that OsANN1 confers abiotic stress tolerance by modulating antioxidant accumulation under abiotic stress. OsANN1-knockdown [RNA interference (RNAi)] plants were more sensitive to heat and drought stresses, whereas OsANN1-overexpression (OE) lines showed improved growth with higher expression of OsANN1 under abiotic stress. Overexpression of OsANN1 promoted SOD (superoxide dismutase) and CAT (catalase) activities, which regulate H2O2 content and redox homeostasis, suggesting the existence of a feedback mechanism between OsANN1 and H2O2 production under abiotic stress. Higher expression of OsANN1 can provide overall cellular protection against abiotic stress-induced damage, and a significant accumulation of OsANN1-green fluorescent protein (GFP) signals was found in the cytosol after heat shock treatment. OsANN1 also has calcium-binding and ATPase activities in vitro, indicating that OsANN1 has multiple functions in rice growth. Furthermore, yeast two-hybrid and bimolecular fluorescence complementation (BiFC) assays demonstrated that OsANN1 interacts with OsCDPK24. This cross-talk may provide additional layers of regulation in the abiotic stress response.

Mapping of quantitative trait locus (QTLs) that contribute to germination and early seedling drought tolerance in the interspecific cross Setaria italica×Setaria viridis.

Drought tolerance is an important breeding target for enhancing the yields of grain crop species in arid and semi-arid regions of the world. Two species of Setaria, domesticated foxtail millet (S. italica) and its wild ancestor green foxtail (S. viridis) are becoming widely adopted as models for functional genomics studies in the Panicoid grasses. In this study, the genomic regions controlling germination and early seedling drought tolerance in Setaria were identified using 190 F7 lines derived from a cross between Yugu1, a S. italica cultivar developed in China, and a wild S. viridis genotype collected from Uzbekistan. Quantitative trait loci were identified which contribute to a number of traits including promptness index, radical root length, coleoptile length and lateral root number at germinating stage and seedling survival rate was characterized by the ability of desiccated seedlings to revive after rehydration. A genetic map with 128 SSR markers which spans 1293.9 cM with an average of 14 markers per linkage group of the 9 linkage groups was constructed. A total of eighteen QTLs were detected which included nine that explained over 10% of the phenotypic variance for a given trait. Both the wild green foxtail genotype and the foxtail millet cultivar contributed the favorite alleles for traits detected in this trial, indicating that wild Setaria viridis populations may serve as a reservoir for novel stress tolerance alleles which could be employed in foxtail millet breeding.

Hyperpolization-activated Ca(2+) channels in guard cell plasma membrane are involved in extracellular ATP-promoted stomatal opening in Vicia faba.

Extracellular ATP (eATP) plays essential roles in plant growth, development, and stress tolerance. Extracellular ATP-regulated stomatal movement of Arabidopsis thaliana has been reported. Here, ATP was found to promote stomatal opening of Vicia faba in a dose-dependent manner. Three weakly hydrolysable ATP analogs (adenosine 5'-O-(3-thio) triphosphate (ATPγS), 3'-O-(4-benzoyl) benzoyl adenosine 5'-triphosphate (Bz-ATP) and 2-methylthio-adenosine 5'-triphosphate (2meATP)) showed similar effects, indicating that ATP acts as a signal molecule rather than an energy charger. ADP promoted stomatal opening, while AMP and adenosine did not affect stomatal movement. An ATP-promoted stomatal opening was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI), the reductant dithiothreitol (DTT) or the Ca(2+) channel blockers GdCl3 and LaCl3. A hyperpolarization-activated Ca(2+) channel was detected in plasma membrane of guard cell protoplast. Extracellular ATP and weakly hydrolyzable ATP analogs activated this Ca(2+) channel significantly. Extracellular ATP-promoted Ca(2+) channel activation was markedly inhibited by DPI or DTT. These results indicated that eATP may promote stomatal opening via reactive oxygen species that regulate guard cell plasma membrane Ca(2+) channels.

Lacking chloroplasts in guard cells of crumpled leaf attenuates stomatal opening: both guard cell chloroplasts and mesophyll contribute to guard cell ATP levels.

Controversies regarding the function of guard cell chloroplasts and the contribution of mesophyll in stomatal movements have persisted for several decades. Here, by comparing the stomatal opening of guard cells with (crl-ch) or without chloroplasts (crl-no ch) in one epidermis of crl (crumpled leaf) mutant in Arabidopsis, we showed that stomatal apertures of crl-no ch were approximately 65-70% those of crl-ch and approximately 50-60% those of wild type. The weakened stomatal opening in crl-no ch could be partially restored by imposing lower extracellular pH. Correspondingly, the external pH changes and K(+) accumulations following fusicoccin (FC) treatment were greatly reduced in the guard cells of crl-no ch compared with crl-ch and wild type. Determination of the relative ATP levels in individual cells showed that crl-no ch guard cells contained considerably lower levels of ATP than did crl-ch and wild type after 2 h of white light illumination. In addition, guard cell ATP levels were lower in the epidermis than in leaves, which is consistent with the observed weaker stomatal opening response to white light in the epidermis than in leaves. These results provide evidence that both guard cell chloroplasts and mesophyll contribute to the ATP source for H(+) extrusion by guard cells.