Mirrorless MEMS imaging: a nonlinear vibrational approach utilizing aerosol-jetted PZT-actuated fiber MEMS scanner for microscale illumination.

This study introduces a novel image capture and lighting techniques using a cutting-edge hybrid MEMS scanner system designed for compact microscopic imaging. The scanner comprises a tapered optical fiber waveguide and innovative aerosol-jet printed PZT (lead zirconate titanate) bimorph push-pull actuators on a stainless-steel substrate, effectively addressing issues that are commonly associated with PZT on silicon substrates such as fracture and layer separation. By leveraging nonlinear vibration, the scanner achieves a spiral scan pattern from a single signal input, in addition to the expected two-dimensional scanning and target illumination from two phase-shifted inputs. This capability is further enhanced by a novel process to taper the optical fiber, which reduces illumination scattering and tunes the fiber to the resonant frequencies of the scanner. The precisely tapered tip enables large fields of view while maintaining independent 2-axis scanning through one-degree-of-freedom actuation. Experimental validation showcases the successful generation of a spiral scan pattern with a 60 µm diameter scan area and a 10 Hz frame rate, effectively reconstructing scanned images of 5 µm lines, cross patterns (15 µm in length with a 5 µm gap), and structures of a Psychodidae wing.

Arabidopsis PDE1 confers phosphate-deficiency tolerance in primary root growth.

KEY MESSAGE: PDE1 acts as a mediator of primary root growth in response to Pi deficiency. Phosphorus is commonly considered as a limiting nutrient for plant growth, which is mainly due to the immobility and uneven distribution of phosphate (Pi) in soils so that available Pi is not adequate in the rhizosphere. Although various mediators have been identified in Pi sensing and response, more details need to be uncovered in plant Pi-deficiency tolerance. Here, we isolated a mutant, termed pde1 (phosphate-deficiency sensitive 1), showing the hypersensitive Pi-deficiency-induced growth inhibition of primary roots. PDE1 encodes a hydroxyphenylpyruvate reductase with rare activity in vitro and repressed by Pi deficiency. Histochemical analysis displayed that Pi-deprived pde1 accumulated more Fe and reactive oxygen species (ROS) in primary roots than the wild type (WT). Addition of ferrozine, a Fe2+ chelator, or a ROS scavenger (e.g., thiourea and potassium iodide), alleviated the sensitivity of Pi-deficiency in pde1 primary roots. By contrast, pde1 showed reduced cotyledon expansion rate with treatment of H2O2 compared to WT. Taken together, these results suggested that PDE1 is responsible for regulating primary root growth in response to Pi deficiency, which is associated with ROS.

UBC6, a ubiquitin-conjugating enzyme, participates in secondary cell wall thickening in the inflorescence stem of Arabidopsis.

Secondary cell wall (SCW) thickening in plant inflorescence stems is a complicated cellular process that is essential for stem strength and biomass. Although Arabidopsis NAC transcription factor (TF) 1 (NST1) regulates the SCW thickening in anther walls, the single T-DNA-insertion mutant (nst1) does not show disrupted SCW thickening in anther endothecium, interfascicular fibers or xylem. To better understand the regulatory mechanism of this process, we generated an ethyl methanesulfonate (EMS)-mutagenized Arabidopsis population with the nst1 background. scd5 (SCW-defective mutant 5) was isolated in a forward genetic screen from the EMS mutant library, which displayed not only less lignin deposition in the interfascicular fiber and xylem than the wild type but also a pendent inflorescence stem. The EMS-induced mutation associated with the scd5 phenotype was found in the 5th exon of At2G46030 that encodes a ubiquitin-conjugating enzyme (UBC6), we thereby renamed the allele nst1 ubc6. Overexpressing UBC6 in nst1 ubc6 rescued the defective SCW, whereas disrupting UBC6 in nst1 by the CRISPR/Cas9 system caused a phenotype similar to that observed in nst1 ubc6. UBC6 was localized to the nucleus and plasma membrane, and possessed E2 ubiquitin-conjugating activity in vitro. MYB7 and MYB32 are considered as transcription repressors in the phenylpropanoid pathway and are involved in NAC TF-related transcriptional regulation in SCW thickening. UBC6 can interact with MYB7 and MYB32 and positively mediate the degradation of MYB7 and MYB32 by the 26S proteasome. Overall, these results indicated the contribution of UBC6 to SCW thickening in Arabidopsis inflorescence stems.

CaRH57, a RNA helicase, contributes pepper tolerance to heat stress.

RNA helicases (RHs) are required for most aspects of RNA metabolism and play an important role in plant stress tolerance. Heat stress (HS) causes the deleterious effects on plant cells, such as membrane disruption and protein misfolding, which results in the inhibition of plant growth and development. In this study, CaRH57 was identified from pepper (Capsicum annuum) and encodes a DEAD-box RH. CaRH57 was induced by HS, and overexpression of CaRH57 in Atrh57-1 rescued the glucose-sensitive phenotype of Atrh57-1, suggesting the functional replacement of CaRH57 to AtRH57. The nucleolus-localized CaRH57 possessed a RH activity in vitro. CaRH57 knockdown impaired pepper heat tolerance, showing severe necrosis and enhanced ROS accumulation in the region of the shoot tip. Additionally, accumulation of aberrant-spliced CaHSFA1d and CaHSFA9d was enhanced, and the corresponding mature mRNA levels were reduced in the TRV2 (Tobacco rattle virus)-CaRH57-infected plants compared with the control plants under HS. Overall, these results suggested that CaRH57 acted as a RH to confer pepper heat tolerance and was required for the proper pre-mRNA splicing of some HS-related genes.

IRR1 contributes to de novo root regeneration from Arabidopsis thaliana leaf explants.

Plants are capable of regenerating adventitious roots (ARs), which is important for plant response to stress and survival. Although great advances in understanding AR formation of leaf explants have been made, the regulatory mechanisms of AR formation still need to be investigated. In this study, irr1-1 (impaired root regeneration) was isolated with the inhibition of adventitious rooting from Arabidopsis leaf explants. The ß-glucuronidase (GUS) signals of IRR1pro::GUS in detached leaves could be detected at 2-6 days after culture. IRR1 is annotated to encode a Class III peroxidase localized in the cell wall. The total peroxidase (POD) activity of irr1 mutants was significantly lower than that of the wild type. Detached leaves of irr1 mutants showed enhanced reactive oxygen species (ROS) accumulation 4 days after leaves were excised from seedlings. Moreover, thiourea, a ROS scavenger, was able to rescue the adventitious rooting rate in leaf explants of irr1 mutants. Addition of 0.1 µM indole-3-acetic acid (IAA) improved the adventitious rooting from leaf explants of irr1 mutants. Taken together, these results indicated that IRR1 was involved in AR formation of leaf explants, which was associated with ROS homeostasis to some extent.

PtoMPO1, a negative mediator, functions in poplar drought tolerance.

Drought, as one of the most severe abiotic stresses in nature, adversely affects plant growth and development. Poplar is a woody plant which is prone to water-deficit sensitivity. Therefore, it is important to improve our understanding of how poplar responds to drought stress. Here, we cloned a gene from Populus tomentosa, namely PtoMPO1. PtoMPO1 encodes a DUF962 domain protein that is a homolog of yeast dioxygenase Mpo1 and Arabidopsis MHP1. The transcripts of PtoMPO1 were repressed by drought stress and ABA. Atmhp1-1 was a T-DNA insertion mutant lacking AtMHP1, and heteroexpression of PtoMPO1 in Atmhp1-1 significantly alleviated the sensitivity of Atmhp1-1 to ABA and NaCl, implying the functional replacement of PtoMPO1 to AtMHP1. PtoMPO1 overexpression decreased but PtoMPO1 mutation enhanced poplar drought tolerance. Furthermore, the expression of drought-related gene PtoRD26 is markedly lower in PtoMPO1-overexpressing plants and notably higher in Ptompo1 mutants compared to that in the wild type. Overall, these results suggested that PtoMPO1 functions as a novel negative mediator for drought tolerance in poplar.

Arabidopsis mitochondrial single-stranded DNA-binding proteins SSB1 and SSB2 are essential regulators of mtDNA replication and homologous recombination.

Faithful DNA replication is one of the most essential processes in almost all living organisms. However, the proteins responsible for organellar DNA replication are still largely unknown in plants. Here, we show that the two mitochondrion-targeted single-stranded DNA-binding (SSB) proteins SSB1 and SSB2 directly interact with each other and act as key factors for mitochondrial DNA (mtDNA) maintenance, as their single or double loss-of-function mutants exhibit severe germination delay and growth retardation. The mtDNA levels in mutants lacking SSB1 and/or SSB2 function were two- to four-fold higher than in the wild-type (WT), revealing a negative role for SSB1/2 in regulating mtDNA replication. Genetic analysis indicated that SSB1 functions upstream of mitochondrial DNA POLYMERASE IA (POLIA) or POLIB in mtDNA replication, as mutation in either gene restored the high mtDNA copy number of the ssb1-1 mutant back to WT levels. In addition, SSB1 and SSB2 also participate in mitochondrial genome maintenance by influencing mtDNA homologous recombination (HR). Additional genetic analysis suggested that SSB1 functions upstream of ORGANELLAR SINGLE-STRANDED DNA-BINDING PROTEIN1 (OSB1) during mtDNA replication, while SSB1 may act downstream of OSB1 and MUTS HOMOLOG1 for mtDNA HR. Overall, our results yield new insights into the roles of the plant mitochondrion-targeted SSB proteins and OSB1 in maintaining mtDNA stability via affecting DNA replication and DNA HR.

Capsicum SIZ1 contributes to ABA-induced SUMOylation in pepper.

Abiotic and biotic stresses are the major factors limiting plant growth. Arabidopsis E3 SUMO ligase SIZ1 plays an essential role in plant stress tolerance. Herein, we identified a SIZ/PAIS-type protein in pepper (Capsicum annuum), namely CaSIZ1, which shares 60 % sequence identity with AtSIZ1. The stems and flowers of pepper had a relatively higher expression of CaSIZ1 than the fruits, leaves, and roots. ABA and NaCl treatments induced CaSIZ1. CaSIZ1 protein was localized in the nucleus and partially rescued the dwarf and ABA-sensitive phenotypes of Atsiz1-2, suggesting the functional replacement of CaSIZ1 with AtSIZ1. We found that CaSIZ1 interacted with CaABI5, and ABA promoted the accumulation of SUMO conjugates in pepper. CaSIZ1 knockdown did not only reduce ABA-induced SUMOylation, but also attenuated the salt tolerance of pepper. Overall, the results of this study suggest that CaSIZ1 has a significant role in ABA-induced SUMOylation and stress response.

Sarracenia purpurea glycerol-3-phosphate acyltransferase 5 confers plant tolerance to high humidity in Arabidopsis thaliana.

Suberin, as a lipid polyester barrier, limits the movement of gas, water, and solutes, and plays important roles in plant protection and growth. In this study, a CDS encoding glycerol-3-phosphate acyltransferase 5 (GPAT5) was cloned from Sarracenia purpurea to investigate the gene function. SpGPAT5 shares 72% identity and 80% similarity to AtGPAT5 that is required for suberin synthesis. Fluorol Yellow 088 staining showed that the S. purpurea pitcher (specific leaf) tube contained more suberin in the adaxial surface compared to the lid, and SpGPAT5 transcripts were detected in the pitcher. Previous reported Atgpat5-1 phenotypes were complemented with SpGPAT5 showing that the Atgpat5-1 seed coat had increased permeability of tetrazolium red and the mutant was sensitive to salt. We also found that SpGPAT5 was able to revert the hyperhydric phenotype of Atgpat5-1 under high humidity. Thus, this study suggests that SpGPAT5 can functionally replace AtGPAT5 and contributes to plant tolerance to high humidity, which maybe assist in understanding the role of suberin-associated waxes in S. purpurea pitchers for water retention.

Arabidopsis SSB1, a Mitochondrial Single-Stranded DNA-Binding Protein, is Involved in ABA Response and Mitochondrial RNA Splicing.

A mitochondrion is a semiautonomous organelle that provides energy for life activities and balances plant growth and stress responses. Abscisic acid (ABA) regulates multiple physiological processes, including seed maturation, seed dormancy, stomatal closure and various abiotic stress responses. However, the relationship between mitochondrial activity and the ABA response is unclear. In this study, an Arabidopsis mutant, ssb1-1, was isolated because of its hypersensitivity toward ABA. Assessment results showed that ABA negatively regulates the expression of Arabidopsis SSB1. Mutations in ABA-insensitive 4 (ABI4) and ABI5, genes of key transcription factors involved in ABA-dependent seed dormancy, attenuated the ABA sensitivity of ssb1-1 during germination, suggesting that Arabidopsis SSB1 may act as a regulator in ABA response. Inhibition of endogenous ABA biosynthesis reversed the NaCl-sensitive phenotype of the ssb1-1 mutant, indicating that enhanced ABA biosynthesis is critical for the salinity stress response of ssb1-1. Moreover, compared to that of the wild type, ssb1-1 accumulated more reactive oxygen species (ROS) and exhibited increased sensitivity to the application of exogenous H2O2 during seed germination. SSB1 is also required for mitochondrial RNA splicing, as indicated by the result showing that SSB1 loss of function led to a decreased splicing efficiency of nad1 intron1 and nad2 intron1. Taken together, our data reported here provide insights into a novel role of Arabidopsis SSB1 in ABA signaling and mitochondrial RNA splicing.

Arabidopsis MHP1, a homologue of yeast Mpo1, is involved in ABA signaling.

Sphingolipids and their intermediates play multiple roles in biological processes. The sphingoid long-chain base component of sphingolipids has emerged as a participant in the regulation of plant biotic and abiotic stress responses. The phytohormone abscisic acid (ABA) regulates many stress responses in plants for environmental adaptation. However, the relationship between the sphingoid bases and ABA is undetermined. In this study, mhp1-1 (the yeast Mpo1 homolog in plants) was isolated through a sodium chloride (NaCl)-sensitivity screen of Arabidopsis transfer DNA (T-DNA) insertion mutants. mhp1-1 was hypersensitivity to salt/osmotic stress and ABA. MHP1 encodes a protein with a domain of unknown function 962 (DUF962). Endoplasmic reticulum-localized MHP1 was found to interact with ABI1. MHP1, a homolog of yeast dioxygenase Mpo1, rescued the growth arrest of mpo1Δ cells caused by ER stress, suggesting functional homology of MHP1 to Mpo1. Overall, MHP1 plays important roles in response to ABA.

SIZ1-Mediated SUMOylation of ROS1 Enhances Its Stability and Positively Regulates Active DNA Demethylation in Arabidopsis.

The 5-methylcytosine DNA glycosylase/lyase REPRESSOR OF SILENCING 1 (ROS1)-mediated active DNA demethylation is critical for shaping the genomic DNA methylation landscape in Arabidopsis. Whether and how the stability of ROS1 may be regulated by post-translational modifications is unknown. Using a methylation-sensitive PCR (CHOP-PCR)-based forward genetic screen for Arabidopsis DNA hyper-methylation mutants, we identified the SUMO E3 ligase SIZ1 as a critical regulator of active DNA demethylation. Dysfunction of SIZ1 leads to hyper-methylation at approximately 1000 genomic regions. SIZ1 physically interacts with ROS1 and mediates the SUMOylation of ROS1. The SUMOylation of ROS1 is reduced in siz1 mutant plants. Compared with that in wild-type plants, the protein level of ROS1 is significantly decreased, whereas there is an increased level of ROS1 transcripts in siz1 mutant plants. Our results suggest that SIZ1-mediated SUMOylation of ROS1 promotes its stability and positively regulates active DNA demethylation.

Arabidopsis glucose-sensitive mutant 3 affects ABA biosynthesis and sensitivity during early seedling development.

In plants, glucose (Glc) plays pivotal roles in development and stress responses mainly by supplying fuel for growth and regulating expression of genes essential for crosstalk with hormonal, oxidative, and defense signaling. However, the complicated relationship between Glc and plant hormones is still not very clear. In this study, gsm3 (glucose-sensitive mutant 3), an Arabidopsis mutant with Glc-sensitive phenotype, was identified. Compared to wild type, the cotyledon expansion rate of gsm3 was significantly decreased under the condition of 4.5% Glc. Fluridone was able to rescue the Glc-induced defects of gsm3 in cotyledon expansion. AAO3 and ABI4 are key genes involved in abscisic acid (ABA) biosynthesis and signaling transduction, respectively. We found that inactivation of AAO3 or ABI4 in gsm3 background led to reduced sensitivity to Glc. These results indicated that increased ABA synthesis resulted in the sensitivity of gsm3 to Glc. Moreover, our results indicated that gsm3 mutant accumulated more ROS, which made it more sensitive to the application of exogenous H2O2. Overall, GSM3 plays an important role in Glc-ABA signaling cascade during seed germination and early seedling growth.

GSM2, a transaldolase, contributes to reactive oxygen species homeostasis in Arabidopsis.

Plants are exposed to various environmental cues that lead to reactive oxygen species (ROS) accumulation. ROS production and detoxification are tightly regulated to maintain balance. Although studies of glucose (Glc) are always accompanied by ROS in animals, the role of Glc in respect of ROS in plants is unclear. We isolated gsm2 (Glc-hypersensitive mutant 2), a mutant with a notably chlorotic-cotyledon phenotype. The chloroplast-localized GSM2 was characterized as a transaldolase in the pentose phosphate pathway. With 3% Glc treatment, fewer or no thylakoids were observed in gsm2 cotyledon chloroplasts than in wild-type cotyledon chloroplasts, suggesting that GSM2 is required for chloroplast protection under stress. gsm2 also showed evaluated accumulation of ROS with 3% Glc treatment and was more sensitive to exogenous H2O2 than the wild type. Gene expression analysis of the antioxidant enzymes in gsm2 revealed that chloroplast damage to gsm2 cotyledons results from the accumulation of excessive ROS in response to Glc. Moreover, the addition of diphenyleneiodonium chloride or phenylalanine can rescue Glc-induced chlorosis in gsm2 cotyledons. This work suggests that GSM2 functions to maintain ROS balance in response to Glc during early seedling growth and sheds light on the relationship between Glc, the pentose phosphate pathway and ROS.

Arabidopsis GSM1 is involved in ABI4-regulated ABA signaling under high-glucose condition in early seedling growth.

In plants, sugar acts as an essential signaling molecule that modulates various aspects of metabolism, growth and development, which are also controlled by phytohormones. However, the molecular mechanism of cross-talk between sugar and phytohormones still remains to be elucidated. We have identified gsm1 (glucose-hypersensitive mutant 1) as a mutant with impaired cotyledon development that shows sensitivity to exogenous abscisic acid (ABA). The addition of fluridone can reverse the glucose (Glc) inhibitory effect in gsm1, implying that endogenous ABA is involved in the Glc response of gsm1. In 4.5% Glc, the expression of Glc-induced ABA-responsive genes in gsm1-1 was nearly two times higher than that in the wild type. Compared to gsm1-1, the gsm1-1 abi4-1 double mutant exhibited reduced sensitivity to Glc and ABA, which was similar to the Glc and ABA insensitive phenotype of abi4-1, suggesting that ABI4 is epistatic to GSM1. In the treatment with 4.5% Glc, the GSM1 transcript level was greatly increased in abi4-1 by almost 4-fold of that in the wild type. These data suggest that GSM1 plays an important role in the ABI4-regulated Glc-ABA signaling cascade during Arabidopsis early seedling growth.

A WD40 protein, AtGHS40, negatively modulates abscisic acid degrading and signaling genes during seedling growth under high glucose conditions.

The Arabidopsis thaliana T-DNA insertion mutant glucose hypersensitive (ghs) 40-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The ghs40-1 mutant displayed severely impaired cotyledon greening and expansion and showed enhanced reduction in hypocotyl elongation of dark-grown seedlings when grown in Glc concentrations higher than 3 %. The Glc-hypersensitivity of ghs40-1 was correlated with the hyposensitive phenotype of 35S::AtGHS40 seedlings. The phenotypes of ghs40-1 were recovered by complementation with 35S::AtGHS40. The AtGHS40 (At5g11240) gene encodes a WD40 protein localized primarily in the nucleus and nucleolus using transient expression of AtGHS40-mRFP in onion cells and of AtGHS40-EGFP and EGFP-AtGHS40 in Arabidopsis protoplasts. The ABA biosynthesis inhibitor fluridone extensively rescued Glc-mediated growth arrest. Quantitative real time-PCR analysis showed that AtGHS40 was involved in the control of Glc-responsive genes. AtGHS40 acts downstream of HXK1 and is activated by ABI4 while ABI4 expression is negatively modulated by AtGHS40 in the Glc signaling network. However, AtGHS40 may not affect ABI1 and SnRK2.6 gene expression. Given that AtGHS40 inhibited ABA degrading and signaling gene expression levels under high Glc conditions, a new circuit of fine-tuning modulation by which ABA and ABA signaling gene expression are modulated in balance, occurred in plants. Thus, AtGHS40 may play a role in ABA-mediated Glc signaling during early seedling development. The biochemical function of AtGHS40 is also discussed.

Characterization of a lily anther-specific gene encoding cytoskeleton-binding glycoproteins and overexpression of the gene causes severe inhibition of pollen tube growth.

This work characterizes an anther/pollen-specific gene that encodes potential intermediate filament (IF)-binding glycoproteins in lily (Lilium longiflorum Thunb. cv. Snow Queen) anthers during the development and pollen germination. LLP13 is a single gene that encodes a polypeptide of 807 amino acids, and a calculated molecular mass of 91 kDa. The protein contains a predicted transmembrane domain at the N-terminus and a conserved domain of unknown function (DUF)593 at the C-terminal half of the polypeptide. Sequence analysis revealed that LLP13 shares significant identity (37-41 %) with two intermediate filament antigen-binding proteins, representing a unique subgroup of DUF593 domain proteins from known rice and Arabidopsis species. The expression of LLP13 gene is anther-specific, and the transcript accumulates only at the stage of pollen maturation. Both premature drying and abscisic acid (ABA) treatment of developing pollen indicated that LLP13 was not induced by desiccation and ABA, but by other developmental cues. Antiserum was raised against the overexpressed LLP13C fragment of the protein in Escherichia coli and affinity-purified antibodies were prepared. Immunoblot analyses revealed that the LLP13 protein was a heterogeneous, anther-specific glycoprotein that accumulated only at the stage of pollen maturation. The protein is not heat-soluble. The level of LLP13 protein remained for 24 h during germination in vitro. Overexpression of LLP13-GFP or GFP-LLP13 in lily pollen tubes caused severe inhibition of tube elongation. The LLP13 protein codistributed with mTalin in growing tubes, suggesting that it apparently decorates actin cytoskeleton and is likely a cytoskeleton-binding protein that binds with IFs that potentially exist in pollen tubes.

AtRH57, a DEAD-box RNA helicase, is involved in feedback inhibition of glucose-mediated abscisic acid accumulation during seedling development and additively affects pre-ribosomal RNA processing with high glucose.

The Arabidopsis thaliana T-DNA insertion mutant rh57-1 exhibited hypersensitivity to glucose (Glc) and abscisic acid (ABA). The other two rh57 mutants also showed Glc hypersensitivity similar to rh57-1, strongly suggesting that the Glc-hypersensitive feature of these mutants results from mutation of AtRH57. rh57-1 and rh57-3 displayed severely impaired seedling growth when grown in Glc concentrations higher than 3%. The gene, AtRH57 (At3g09720), was expressed in all Arabidopsis organs and its transcript was significantly induced by ABA, high Glc and salt. The new AtRH57 belongs to class II DEAD-box RNA helicase gene family. Transient expression of AtRH57-EGFP (enhanced green fluorescent protein) in onion cells indicated that AtRH57 was localized in the nucleus and nucleolus. Purified AtRH57-His protein was shown to unwind double-stranded RNA independent of ATP in vitro. The ABA biosynthesis inhibitor fluridone profoundly redeemed seedling growth arrest mediated by sugar. rh57-1 showed increased ABA levels when exposed to high Glc. Quantitative real time polymerase chain reaction analysis showed that AtRH57 acts in a signaling network downstream of HXK1. A feedback inhibition of ABA accumulation mediated by AtRH57 exists within the sugar-mediated ABA signaling. AtRH57 mutation and high Glc conditions additively caused a severe defect in small ribosomal subunit formation. The accumulation of abnormal pre-rRNA and resistance to protein synthesis-related antibiotics were observed in rh57 mutants and in the wild-type Col-0 under high Glc conditions. These results suggested that AtRH57 plays an important role in rRNA biogenesis in Arabidopsis and participates in response to sugar involving Glc- and ABA signaling during germination and seedling growth.

New insights into desiccation-associated gene regulation by Lilium longiflorum ASR during pollen maturation and in transgenic Arabidopsis.

LLA23, a member of the abscisic acid-, stress-, and ripening-induced (ASR) protein family, was previously isolated from lily (Lilium longiflorum) pollen. The lily ASR is induced through desiccation-associated ABA signaling transduction in the pollen. ASRs are highly hydrophilic and intrinsically unstructured proteins with molecular masses generally less than 18 kDa. LLA23 is abundant in the cytoplasm and nuclei of both vegetative and generative cells of pollen grains. The protein in the nucleus and in the cytoplasm is partly regulated by dehydration. A dual role is proposed for LLA23, as a regulator and a protective molecule, upon exposure to water deficits. This chapter reviews the current state of literature on Asr genes, protein structure, function, and their responses to various stresses. In a study, a genome-wide microarray was used to monitor the expression of LLA23-regulated genes, focusing on the relationship between ASR-, glucose-, and drought-inducible genes, and outlined the difference and cross talk of gene expression among these signaling networks. A strong association was observed in the expression of stress-responsive genes and found 25 genes that respond to all three treatments. Highly inducible genes were also found in each specific stress treatment. Promoter sequence analysis of LLA23-inducible genes enabled us not only to identify possible known cis-acting elements in the promoter regions but also to expect the existence of novel cis-acting elements involved in ASR-responsive gene expression. ASR can be used to improve crops and economically important plants against various environmental stresses.