Expression of an activated rasD gene changes cell fate decisions during Dictyostelium development.

It has been previously demonstrated that the expression of an activated rasD gene in wild-type Dictyostelium cells results in formation of aggregates with multitips, instead of the normal single tips, and a block in further development. In an attempt to better understand the role of activated RasD development, we examined cell-type-specific gene expression in a strain stably expressing high levels of RasD[G12T]. We found that the expression of prestalk cell-specific genes ecmA and tagB was markedly enhanced, whereas the expression of the prespore cell-specific gene cotC was reduced to very low levels. When the fate of cells in the multitipped aggregate was monitored with an ecmA/lacZ fusion, it appeared that most of the cells eventually adopted prestalk gene expression characteristics. When mixtures of the [G12T]rasD cells and Ax3 cells were induced to differentiate, chimeric pseudoplasmodia were not formed. Thus, although the [G12T]rasD transformant had a marked propensity to form prestalk cells, it could not supply the prestalk cell population when mixed with wild-type cells. Both stalk and spore cell formation occurred in low cell density monolayers of the [G12T]rasD strain, suggesting that at least part of the inhibition of stalk and spore formation during multicellular development involved inhibitory cell interactions within the cell mass. Models for the possible role of rasD in development are discussed.     

Mutational Analysis of the Role of Rap1 in Regulating Cytoskeletal Function inDictyostelium

It was shown previously that increased expression of theras-relatedrap1gene inDictyostelium discoideumaltered cell morphology (Rebsteinet al., Dev. Genet.,1993, 14, 347–355). Vegetative Rap1 transformants were more flattened and spread than parental Ax2 cells and had increased F-actin near the cell periphery. In addition, starving Rap1 cells were inhibited in the rapid cell contraction that occurs upon refeeding with nutrient media. In this communication, we show that expression of Rap also markedly reduces the contraction response that occurs upon addition of azide to vegetative cells. The changes in cell morphology, the refeeding contraction response, and the azide contraction response have been used to analyze mutants of Rap1 generated by site-directed mutagenesis. The substitution G12V, predicted to increase the proportion of protein binding GTP, did not alter the effect of Rap on cell morphology or on its ability to inhibit the contraction response to azide, but modestly enhanced the ability of Rap1 to inhibit cell rounding in response to nutrient media. The substitution S17N, predicted to restrict the protein to the GDP-bound state, did not produce the flattened cell morphology and abolished the inhibitory effects of Rap in the two cell contraction assays. These results are consistent with a requirement of GTP binding for the Rap-induced effects. Transformants carrying the Rap-S17N protein had a more polar morphology than the parental Ax2 cells, suggesting the possibility that Rap-S17N interferes with the ability of endogenous Rap to regulate the cytoskeleton. Substitutions at amino acid 38, within the presumptive effector domain, reduced but did not abolish the effects of Rap1 on cell contraction, while the substitution T61Q had no effect on Rap1 activity. Taken together, the results suggest that Rap may have multiple regulatory effects on cytoskeletal function.     

Theste13+ gene encoding a putative RNA helicase is essential for nitrogen starvation-induced G1 arrest and initiation of sexual development in the fission yeastSchizosaccharomyces pombe

When the fission yeastSchizosaccharomyces pombe is starved for nitrogen, the cells are arrested in the G1 phase, enter the G0 phase and initiate sexual development. Theste13 mutant, however, fails to undergo a G1 arrest when starved for nitrogen and since this mutant phenotype is not suppressed by a mutation in adenylyl cyclase (cyr1), it would appear thatste13 ⁺ either acts independently of the decrease in the cellular cAMP level induced by starvation for nitrogen, or functions downstream of this controlling event. We have used functional complementation to clone theste13 ⁺ gene from anS. pombe genomic library and show that its disruption is not lethal, indicating that, while the gene is required for sexual development, it is not essential for cell growth. Nucleotide sequencing predicts thatste13 ⁺ should encode a protein of 485 amino acids in which the consensus motifs of ATP-dependent RNA helicases of the DEAD box family are completely conserved. Point mutations introduced into these consensus motifs abolished theste13 ⁺ functions. The predicted Ste13 protein is 72% identical to theDrosophila melanogaster Me31B protein over a stretch of 391 amino acids. ME31B is a developmentally regulated gene that is expressed preferentially in the female germline and may be required for oogenesis. Expression of ME31B cDNA inS. pombe suppresses theste13 mutation. These two evolutionarily conserved genes encoding putative RNA helicases may play a pivotal role in sexual development.     

Stalk cell differentiation without polyketides in the cellular slime mold

Polyketides induce prestalk cell differentiation in Dictyostelium. In the double-knockout mutant of the SteelyA and B polyketide synthases, most of the pstA cells—the major part of the prestalk cells—are lost, and we show by whole mount in situ hybridization that expression of prestalk genes is also reduced. Treatment of the double-knockout mutant with the PKS inhibitor cerulenin gave a further reduction, but some pstA cells still remained in the tip region, suggesting the existence of a polyketide-independent subtype of pstA cells. The double-knockout mutant and cerulenin-treated parental Ax2 cells form fruiting bodies with fragile, single-cell layered stalks after cerulenin treatment. Our results indicate that most pstA cells are induced by polyketides, but the pstA cells at the very tip of the slug are induced in some other way. In addition, a fruiting body with a single-cell layered, vacuolated stalk can form without polyketides.     

Rap1a is a key regulator of fibroblast growth factor 2-induced angiogenesis and together with Rap1b controls human endothelial cell functions

Angiogenesis, the formation of new blood vessels from existing vasculature, is regulated primarily by endothelial cell activity. We show herein that the Ras family GTPase Rap1 has a key role in the regulation of angiogenesis by modulating endothelial cell functions. Blood vessel growth into fibroblast growth factor 2 (FGF2)-containing Matrigel plugs was absent from rap1a^−/− mice, and aortic rings derived from rap1a^−/− mice failed to sprout primitive tubes in response to FGF2, when the tissue was embedded in Matrigel. Knocking down either rap1a or rap1b, two closely related rap1 family members, in human microvascular endothelial cells (HMVECs) by utilizing siRNA confirmed that Rap1 plays key roles in endothelial cell function. The rap1a or rap1b knockdown resulted in decreased adhesion to extracellular matrices and impaired cell migration. HMVEC monolayers lacking Rap1 had increased permeability, and Rap1-deficient endothelial cells failed to form three-dimensional tubular structures when they were plated on Matrigel in vitro. Finally, the activation levels of extracellular signal-regulated kinase (ERK), p38, and Rac, which are important signaling molecules in angiogenesis, were all reduced in response to FGF2 when either of the Rap1 proteins was depleted. These observations place Rap1 centrally in the human angiogenic process and suggest that both the Rap1a and Rap1b proteins are required for angiogenesis and that Rap1 is a critical mediator of FGF-induced ERK activation.     

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae.

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.     

The isolation and characterization of a mutant allele at a new X-linked locus,mex, affecting NADP+-dependent enzymes inDrosophila melanogaster

The isolation and characterization of mutant alleles in a regulatory gene affecting NADP⁺-dependent enzymes are described. The locus,mex, is at position 26.5 ± 0.74 on the X chromosome ofDrosophila melanogaster. The newly isolated mutant allele,mex ¹, is recessive to either themex allele found in Oregon-R wild-type individuals or that found in thecm v parental stock in which the new mutants were induced. Themex ¹ mutant allele is associated with statistically significant decreases in malic enzyme (ME) specific activity and ME specific immunologically cross-reacting material (ME-CRM) in newly emerged adult males. During this same developmental stage in males, the NADP⁺-dependent isocitrate dehydrogenase specific activity increases to statistically significant levels. Females of themex ¹ mutant strain show statistically significant elevated levels of the pentose phosphate shunt enzymes, glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. Isoelectric focusing and thermolability comparisons of the active ME from mutant and control organisms indicate that the enzyme is the same. Developmental profiles ofmex ¹ and control strains indicate that this mutant allele differentially modulates the levels of ME enzymatic activity and ME-CRM during development. This work was supported by an Operating Grant from the Natural Sciences and Engineering Research Council of Canada to M.M.B.     

Association between IFNA genotype and the risk of sarcoidosis

Sarcoidosis is known to be a systemic granulomatous disorder characterized by a cell-mediated Th1-type inflammatory response. To identify a key genetic factor in the pathogenesis of sarcoidosis, we investigated single nucleotide polymorphisms within 10 candidate genes involved in type 1 immune process (IFNA17, IFNB, IFNG, IFNGR1, IFNGR2, IL12B, IL12RB1, IL12RB2, ETA-1, and NRAMP1) in an association-based study of 102 Japanese patients with sarcoidosis, 114 with tuberculosis, and 110 control subjects. After correction for multiple testing, an IFNA17 polymorphism (551TG) was found to be associated with susceptibility to sarcoidosis (odds ratio 3.27 [95% CI: 1.44–7.46], P=0.004, P_c=0.04), but not to tuberculosis. We observed no significant associations with the other polymorphisms of the Th1-related genes. We further typed another IFNA polymorphism (IFNA10 60TA) and confirmed two major haplotypes of the IFNA gene, viz., allele 1: IFNA10 [60T]-IFNA17 [551T] and allele 2: IFNA10 [60A]-IFNA17 [551G], in the Japanese population. In healthy subjects, IFNA allele 2, which is over-represented in patients with sarcoidosis, was significantly associated with increased IFN- and IL-12p70 production induced by Sendai virus in vitro. This study suggests that possession of the IFNA allele with higher levels of IFN- significantly increases the risk of sarcoidosis.M. Akahoshi and M. Ishihara contributed equally to this work     

Benzo[a]pyrene-induced cell cycle progression is through ERKs/cyclin D1 pathway and requires the activation of JNKs and p38 mapk in human diploid lung fibroblasts

Treatment of cells with carcinogen Benzo[a]pyrene (B[a]P) allows cells to evade G1 arrest and induces cells abnormal proliferation. However, the mechanisms of its action at cellular level are not well understood. To address this question, normal human embryo lung diploid fibroblasts (HELF) were selected in the present study. We found that exposure of cells with 2.5 μM of B[a]P for 24 h resulted in a decrease of G1 population by 11.9% (P < 0.05) and a increase of S population by 17.2% (P < 0.05). Treatment of cells with B[a]P also caused dose-related activation of MAPK and induction of cyclin D1 protein expression, whereas the CDK4 protein levels were not significantly affected by B[a]P. Overexpression of cyclin D1 protein stimulated by B[a]P was significantly inhibited by 50 μM AG126 (an inhibitor of ERK1/2), but not by 25 μM SP600125 (an inhibitor of JNK1/2) or 5 μM SB203580 (an inhibitor of p38 mapk), suggesting that B[a]P-induced cyclin D1 expression was only regulated by ERK1/2 pathway. However, AG126, SP600125 or SB203580 led to cell cycle significantly arrested in G1 phase, indicating that ERK1/2, JNK1/2 and p38 mapk pathways are all required for B[a]P-induced G1/S transition. In addition, HELF cells transfecting with antisense cyclin D1 cDNA or antisense CDK4 cDNA showed significantly G1 arrest after B[a]P stimulation. These results suggested that B[a]P exposure accelerated the G1→S transition by activation of MAPK signaling pathways. Cyclin D1 and CDK4 are rate-limiting regulators of the G1→S transition and expression of cyclin D1 is predominantly regulated by ERK1/2 pathway in HELF cells.     

Transient expression of a mitochondrial gene cluster including rps4 is essential for the phase‐shift of Dictyostelium cells from growth to differentiation

Using synchronized Dictyostelium discoideum Ax‐2 cells and the differential display method, a mitochondrial gene cluster (referred to as differentiation‐associated gene 3; dia3) was isolated as one of the genes expressed specifically during the transition of Ax‐2 cells from growth to differentiation. The dia3 gene encodes for a mitochondrial protein cluster (NADH dehydrogenase (NAD) subunit 11, 5, ribosomal protein S4 (RPS4), RPS2, and NAD4L). Northern blot analysis using nonsynchronized Ax‐2 cells has shown that the dia3 RNA of about 8 kb is scarcely expressed during the vegetative growth phase, and the maximal expression was attained at 2 h after starvation. To analyze the gene function of dia3, we tried inactivation of rps4 by means of homologous recombination and obtained several transformed clones showing mitochondrial DNA heteroplasmy. The transformed cells grew normally in nutrient medium, but their development after starvation was greatly impaired, thus resulting in the failure of many cells to differentiate. In this connection, the cAMP receptor 1 (car1) expression, which is one of the earliest markers of differentiation, was found to be markedly reduced in the rps4‐inactivated cells. Dev. Genet. 25:339–352, 1999. © 1999 Wiley‐Liss, Inc.     

Identification of Rap1 as a target for the Crk SH3 domain-binding guanine nucleotide-releasing factor C3G.

C3G, which was identified as a Crk SH3 domain-binding guanine nucleotide-releasing factor, shows sequence similarity to CDC25 and Sos family proteins (S. Tanaka, T. Morishita, Y. Hashimoto, S. Hattori, S. Nakamura, M. Shibuya, K. Matuoka, T. Takenawa, T. Kurata, K. Nagashima, and M. Matsuda, Proc. Natl. Acad. Sci. USA 91:3443-3447, 1994). The substrate specificity of C3G was examined by in vitro and in vivo experiments. C3G markedly stimulated dissociation of bound GDP from Rap1B but marginally affected the same reaction of other Ras family proteins (Ha-Ras, N-Ras, and RalA). C3G also stimulated binding of GTP-gamma S [guanosine 5'-3-O-(thio)triphosphate] to Rap1B. When C3G and Rap1A were expressed in COS7 cells, marked accumulation of the active GTP-bound form of Rap1A was observed, while Sos was not effective in the activation of Rap1A. These results clearly show that C3G is an activator for Rap1. Furthermore, expression of C3G with a membrane localization signal in a v-Ki-ras transformant, DT, induced a reversion of the cells to the flat form, possibly through the activation of endogenous Rap1.     

Precocious sporulation and developmental lethality in yelA null mutants of Dictyostelium

A novel developmental gene, yelA, has been found that plays an essential role in regulating terminal differentiation of Dictyostelium discoideum. Strains in which yelA is disrupted by plasmid insertion are arrested at the tight mound stage but accumulate the bright yellow pigment characteristic of mature sori. Although these mutant strains do not form fruiting bodies, many of the cells encapsulate within the mounds. Sporulation occurs about 6 hours earlier in yelA⁻ cells than in wild-type cells, accompanied by precocious expression of a prespore gene, spiA. However, the spores are defective and lose viability over a period of several hours. Unencapsulated cells also die unless they are dissociated from the mounds and shaken in suspension. The yelA gene was isolated by plasmid rescue and found to encode a protein of 102 kDa in which the N-terminal sequence shows significant similarity to domains found in the eIF-4G subunits of the translational initiation complex eIF-4F. In wild-type cells yelA mRNA first accumulates at 8 hours of development and is maintained in both prespore and prestalk cells until culmination when it is found only is stalk cells. Mutations in yelA can partially suppress the block to sporulation in mutant strains in which either of the prestalk genes tagB or tagC is disrupted such that an encapsulation signal is not produced. It appears that premature encapsulation is normally inhibited by YelA until a signal is received from prestalk cells during culmination. Dev. Genet. 20:307–319, 1997. © 1997 Wiley-Liss, Inc.     

Loss of cAMP-specific phosphodiesterase rescues spore development in G protein mutant in Dictyostelium

Cyclic AMP (cAMP) is an important intracellular signaling molecule for many G protein-mediated signaling pathways but the specificity of cAMP signaling in cells with multiple signaling pathways is not well-understood. In Dictyostelium, at least two different G protein signaling pathways, mediated by the Gα2 and Gα4 subunits, are involved with cAMP accumulation, spore production, and chemotaxis and the stimulation of these pathways results in the activation of ERK2, a mitogen-activated protein kinase that can down regulate the cAMP-specific phosphodiesterase RegA. The regA gene was disrupted in gα2⁻ and gα4⁻ cells to determine if the absence of this phosphodiesterase rescues the development of these G protein mutants as it does for erk2⁻ mutants. The regA⁻ mutation had no major effects on developmental morphology but enriched the distribution of the Gα mutant cells to the prespore/prestalk border in chimeric aggregates. The loss of RegA function had no effect on Gα4-mediated folate chemotaxis. However, the regA gene disruption in gα4⁻ cells, but not in gα2⁻ cells, resulted in a substantial rescue and acceleration of spore production. This rescue in sporulation required cell autonomous signaling because the precocious sporulation could not be induced through intercellular signaling in chimeric aggregates. However, intercellular signals from regA⁻ strains increased the expression of the prestalk gene ecmB and accelerated the vacuolization of stalk cells. Intercellular signaling from the gα4⁻regA⁻ strain did not induce ecmA gene expression indicating cell-type specificity in the promotion of prestalk cell development. regA gene disruption in a Gα4^HC (Gα4 overexpression) strain did not result in precocious sporulation or stalk cell development indicating that elevated Gα4 subunit expression can mask regA⁻ associated phenotypes even when provided with wild-type intercellular signaling. These findings indicate that the Gα2 and Gα4-mediated pathways provide different contributions to the development of spores and stalk cells and that the absence of RegA function can bypass some but not all defects in G protein regulated spore development.     

Altered morphology of vegetative amoebae induced by increased expression of the Dictyostelium discoideum ras-related gene rap1

The rap1 gene of Dictyostelium discoideum is a member of the ras-gene superfamily of low molecular weight GTPase proteins. The rapl gene is expressed both during growth and development in D. discoideum. To examine the action of the Rapl protein in D. discoideum, the rap1 cDNA was expressed under the control of the inducible discoidin promoter. Treatment with conditioned media, which induces the discoidin promoter, increased Rap1 protein levels in vegetative cells approximately six fold. Overexpression of the Rapl protein correlated with the appearance of morphologically aberrant vegetative amoebae: cells were extensively spread and flattened. The distribution of F-actin was altered in these cells, with an increase in actin staining around the cell periphery. Induction of the discoidin promoter by starvation in the rapl transformants also resulted in spread flat cells. When starved D. discoideum amoebae are refed with HL5 media, the cells rapidly respond by rounding up. By contrast, the rapl transformant cells showed a pronounced delay in rounding up. Rapid tyrosine phosphorylation of a p45 protein occurred in both control cells and the rapl transformant upon refeeding, implying that the signal transduction pathway leading to tyrosine phosphorylation remained functional in the rapl transformant. We propose that the Rapl protein functions in the regulation of cell morphology in D. discoideum. © 1993Wiley-Liss, Inc.     

High-level expression of the colicin A lysis protein

Summary Two plasmids that overproduce the colicin A lysis protein, Cal, are described. Plasmid AT1 was constructed by a deletion in the colicin A operon, which placed thecal gene near a truncatedcaa gene in such a way that both gene products were synthesized at high levels following induction. Plasmid Ck4 was constructed by insertion of thecal gene downstream from thetac promoter of an expression vector. Overproduction of Cal was obtained after mitomycin C induction of pAT1 cells and after IPTG induction of pCK4 cells. The kinetics of Cal synthesis were examined with [³⁵S] methionine and [2-³H] glycerol inlpp orlpp ⁺ host strains. Each of the steps of the lipid modification and maturation pathway of Cal was demonstrated. The modified precursor form of overproduced Cal was not chased as efficiently as when it is produced in pColA cells. After treatment with globomycin, a significant amount of this modified precursor form accumulated and was degraded with time into smaller acylated proteins, but without release of the signal peptide. Release of cellular proteins and quasi-lysis were observed after about 1 hour of induction for cells containing either plasmid. In addition, in Cal-overproducing cells, the rate of quasi-lysis was increased but not its extent. InpldA cells, quasi-lysis was reduced but not abolished. Lethality of the Cal induction in the overproducing cells was in the same range as that in wild-type cells.     

The regulation of glycoprotein synthesis by cAMP in Dictyostelium

Dictyostelium discoideum (Dd) 1-H vegetative amoebae exposed to cAMP differentiate into mature stalk cells within 48 h [6]. It was of interest to monitor the patterns of glycoprotein synthesis in the amoebae during the first 5 h of exposure to cAMP and phosphate buffer (PB) controls. Following the exposure period the amoebae were labeled with -[6-³H]fucose. It was determined by both silver grain counts of autoradiographs and scintillation spectroscopy that within minutes cAMP effects an inhibition of [³H]fucose incorporation. However, by 5 h of exposure both experimentals and controls lose a major amount of their labeling capacity based upon the initial PB control value. Vegetative amoebae exposed to cAMP mimics the sparse labeling found in prestalk cells. Prestalk cells synthesize cellulose as a result of cAMP-induced gluconeogenesis and consequently glycoprotein synthesis is reduced. Cellular interactions promoted by cAMP appears to initiate prestalk cell differentiation during the pre-aggregation phase of development. This event is accompanied by a loss in the ability of the aggregating cells to synthesize glycoprotein.     

细胞色素c在盘基网柄菌细胞凋亡中的作用

细胞色素c在细胞凋亡中发挥着重要的作用,其作用机理在高等真核生物及低等真核生物酵母中已经比较清楚,但在盘基网柄菌(Dictyostelium discoideum)中的作用却没有相关报道.所以我们用western blot和实时荧光定量PCR的方法分别测定了盘基网柄菌前柄细胞和前孢子细胞中细胞色素c的含量及表达量的变化...     

Comparative phylogenetic analysis of aquaporins provides insight into the gene family expansion and evolution in plants and their role in drought tolerant and susceptible chickpea cultivars

Aquaporins (AQPs) are water channel proteins that play a significant role in drought stress. Although the AQPs identified in multiple plant species, there is no detailed evolutionary and comparative study of AQPs regarding chickpea plant. The current study involved evolutionary analyses coupled with promoter and expression analyses of chickpea AQPs (CaAQPs). A total of 924 non-redundant AQPs were studied in 24 plant species including algae, mosses, lycophytes, monocots and dicots. Phylogenetic analysis demonstrated a clear divergence of eight AQP subfamilies (LIPs, SIPs, GIPs, NIPs, XIPs, PIPs, HIPs and TIPs). The comparative phylogenetic trees of AQP subfamilies among Arabidopsis, soybean, common bean, maize and chickpea demonstrated that the AQPs were highly species-specific. Interestingly, the dual NPA motif was conserved in all species. However, the ar/R selectivity filter signatures [W/T/S/N/G/A]-[V/S/L/I/A]-[S/G/A]-R (in NIPs), F-H-T-R (in PIPs), [H/N/Q/S]-[A/I/L/S/V]-[A/G]-[A/C/L/M/R/V] (in TIPs) and [V/I/L/M]-[V/I/A/F/M]-[A/S/F/C]-[N/F/L/I/A/S (in SIPs) were found in five species. Moreover, the Froger's positions (P1-P5) were found as [F/L/Y]-[S/T]-A-Y-[L/I/M/V/F] (in NIPs), [Q/E/M]-S-A-F-W (in PIPs), [A/L/S/T/V]-[A/C/N/S/T/V]-[P/R/S]-[Y/N/F]-[W/Q] (in TIPs) and [I/M/F]-[A/V]-[A/V]-Y-W (in SIPs). The MEME motif analyses showed that most of the motifs were specific to subfamily and subgroups. Tissue-specific expression profiling of CaAQPs revealed that CaTIPs and CaPIPs are highly expressed in most of the tissues, while CaNIPs and CaSIPs have low expression. In promoter analysis of CaAQPs, multiple stress-related cis-acting elements e.g. MYB, MYC, ABRE, etc. were found. Semi-quantitative RT-PCR analysis showed that CaPIP2;3 and CaNIP3;1 are positive regulator, while CaSIP1;1 and CaPIP2;1 have a negative role in drought tolerance. The findings and implications of this study are discussed in detail.