Introduction
============

Plants are exposed to various environmental conditions, however, plants can not change their location to avoid unfavorable circumstance ([@B26],[@B27], [@B29],[@B30]). Among multiple stresses, drought stress is one of the most harsh environmental stresses ([@B25]; [@B11]; [@B1]). To date, plants have developed sophisticated strategies to counteract sudden environmental changes. Many secondary messengers, including abscisic acid (ABA), and hydrogen peroxide (H~2~O~2~), are involved in plant stress transduction ([@B25]; [@B37]; [@B8]; [@B5]; [@B23]). Both endogenous concentrations and the underlying signaling pathways of ABA and H~2~O~2~ play essential roles in plant drought stress responses ([@B8]; [@B5]; [@B11]; [@B23]; [@B19]).

*Arabidopsis Low Temperature-Induced 30* (LTI30)/XERO2 belongs to the group II late embryogenes abundant protein (LEA)/dehydrin family ([@B24]). The transcript level of *AtLTI30/XERO2* can be induced by ABA, cold, dehydration, wounding, and salt stresses ([@B34]; [@B24]; [@B20]; [@B2]). Overexpression of *AtLTI30/XERO2* enhances freezing stress resistance in *Arabidopsis* ([@B22]). AtCBF1, AtCBF2, and AtCBF3 (also known as AtDREB1b, AtDREB1c, and AtDREB1a, respectively) are important transcription factors in plant abiotic stress responses. To date, many stress-responsive genes with C-repeat (CRT)/dehydration-responsive element (DRE) in the promoters have been identified as the direct targets of AtCBFs. These genes include *COR* (*cold regulated*), *ERD* (*early responsive to dehydration*), *KIN* (*cold inducible*), *LTI* (*low-temperature induced*), and *RD* (*responsive to dehydration*) ([@B9]; [@B38]; [@B4]; [@B31]). Using multiple combinations of mutations in the promoter of *AtLTI30/XERO2*, [@B2] found that two of the ACGT and DRE/CRT elements in the promoter of *AtLTI30/XERO2* were essential for cold and ABA transcriptional induction of *AtLTI30/XERO2*. [@B18] found that the lysine-rich segment of AtLTI30/XERO2 showed sequence similarity with the animal chaperone heat shock protein 90 (HSP90). The conserved segments of AtLTI30 exerted its biological function more locally upon interaction with specific biological targets. Moreover, [@B7] identified three factors that regulate the lipid interaction of LTI30 *in vitro*, including the pH dependent His on/off switch, reversal of membrane binding by proteolytic digestion, and phosphorylation by protein kinase C.

Although *AtLTI30* has been shown to be involved in plant freezing stress resistance, the other roles of *AtLTI30* and the underlying mechanisms remain unknown. In this study, the expression and function of *AtLTI30* were characterized in response to drought stress treatment. We investigated the effects of manipulation of *AtLTI30* expression on drought stress resistance, as well as the underlying mechanisms. The results indicate that *AtLTI30* is a positive regulator of drought stress resistance in *Arabidopsis*.

Materials and Methods {#s1}
=====================

Plant Materials and Growth Conditions
-------------------------------------

After stratification at 4°C for 3 days in darkness, *Arabidopsis thaliana* seeds were sown in soil in a growth chamber, and watered with a nutrient solution twice per week. The growth chamber was controlled at 23°C, with an irradiance of about 150 μmol quanta m^-2^ s^-1^, under 65% relative humidity and 16-h light and 8-h dark cycles. The *lti30-1* (SALK_114915) and *lti30-2* (SALK_016819) mutants were obtained from the *Arabidopsis* Biological Resource Center (ABRC).

RNA Isolation, Semi-quantitative RT-PCR and Quantitative Real-time PCR
----------------------------------------------------------------------

Total RNA was extracted and purified using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) and RQ1 RNase-free DNase (Promega, Madison, WI, USA). First-strand cDNA was synthesized from total RNA using reverse transcriptase (TOYOBO, Osaka city, Japan) as ([@B26],[@B27], [@B29],[@B30], [@B28]) previously described. Semi-quantitative RT-PCR was performed as [@B26] described using *ubiquitin 10* (*UBQ10*) as the internal control. Quantitative real-time PCR was performed using the CFX96^TM^ Real-Time System (BIO-RAD, Hercules, CA, USA) and the comparative ΔΔCT method with *UBQ10* as a reference gene following ([@B26],[@B27], [@B29],[@B30], [@B28]). The primers of *UBQ10* (At4g05320) and *LTI30* (At3g50970) are shown in Supplementary Table [S1](#SM1){ref-type="supplementary-material"}.

Construction of Vectors and Generation of Transgenic Lines
----------------------------------------------------------

For the *pLTI30::β-glucuronidase* (*GUS*) transgenic construction, the promoter region of *AtLTI30* was amplified by PCR and inserted into the *BamHI* site of the pBI101.2 vector with kanamycin resistance. For *AtLTI30* overexpressing transgenic construction, the coding region of *AtLTI30* was amplified by PCR and inserted into the *SmaI/XhoI* sites of the pBIM vector with kanamycin resistance under the control of the cauliflower mosaic virus (CaMV) 35S promoter ([@B36]). The responsible primers for the above vector constructions are shown in Supplementary Table [S2](#SM1){ref-type="supplementary-material"}. The recombinant constructions were confirmed by DNA sequencing and introduced into wild type (WT) plants of Columbia-0 (Col-0) using *Agrobacterium tumefaciens* strain GV3101-mediated transformation and the floral dip method ([@B3]). Thereafter the transgenic plants were selected on MS medium using kanamycin resistance, and further confirmed by PCR analysis.

GUS Staining and Quantification of GUS Activity
-----------------------------------------------

Glucuronidase staining and quantification of GUS activity were performed as [@B13] previously described. For GUS staining, *proLTI30::GUS* transgenic plants were incubated in staining solution (100 mM sodium phosphate buffer, pH 7.5, 0.5 mM K~3~\[Fe(CN)~6~\], and 0.5 mM K~4~\[Fe(CN)~6~\], 10 mM EDTA, 1.0 mM 5-bromo--chloro-3-indolyl-β-*[D]{.smallcaps}*-glucuronide and 0.1% Triton X-100) at 37°C for 8 h, after which the plants were incubated in 70% ethanol to remove chlorophyll. GUS activity was quantified by detecting the conversion of 4-methylumbelliferone from the substrate 4-methylumbelliferyl-β-glucuronide in the same concentration of protein extract, as [@B13] previously described.

Determination of ABA Sensitivity
--------------------------------

For ABA sensitivity assay, different genotypes of *Arabidopsis* seeds were sterilized with 70% (v/v) ethyl alcohol, 5% (w/v) NaClO, and deionized water. The seeds were stratified at 4°C for 3 days, thereafter were sown on Murashige and Skoog (MS) medium plates containing different concentrations of ABA. Germination ratios as seen with emerged radicals and green cotyledons were scored after 10 days in the growth chamber. Stomatal aperture in *Arabidopsis* leaves was determined as [@B26] described.

Plant Drought Stress Treatment and Drought Stress Resistance Assay
------------------------------------------------------------------

For drought stress, 14-day-old *Arabidopsis* plants in the soil were subjected to control (well-watered) and drought stress (withheld water) conditions for another 21 days. More than three pots of each variety (27 plants) were used in each biological repeat, and all these pots with plants were rotated daily during drought stress to minimize the environment effect. The survival rate of stressed plants was recorded after re-watered for 4 days later than 21 days of drought stress treatment. Relative *in vitro* leaf water loss rate was expressed as percent change in leaf fresh weight (FW) as ([@B26],[@B27], [@B29],[@B30], [@B28]) described.

Quantification of Hydrogen Peroxide (H~2~O~2~) and Catalase (CAT) Activity
--------------------------------------------------------------------------

The concentration of H~2~O~2~ in plant leaves was determined using the titanium sulfate method, and CAT (EC 1.11.1.6) activity was determined using the enzyme assay kit as ([@B26],[@B27], [@B29],[@B30], [@B28]) previously reported.

The Determination of Proline Content
------------------------------------

Quantification of endogenous proline content was performed as [@B26],[@B27]) previously described. Briefly, endogenous proline in plant leaves was extracted using 3% (w/v) sulfosalicylic acid, and the red solution at the absorbance of 520 nm was determined by adding the mixtures of ninhydrin reagent and glacial acetic acid to the extractions.

Statistical Analysis
--------------------

All experiments were performed with at least three biological repeats, and plant leave samples in each biological repeat were mixture samples of at least 10 plants per genotype. Student's *t*-test and analysis of variance (AVOVA) were used to analysis the significant difference, and asterisk symbols (^∗^) indicate the significant differences of *p* \< 0.05 in comparison to WT.

Results
=======

The Expression Pattern of *AtLTI30*
-----------------------------------

Using *proLTI30::GUS* transgenic plants, we found that *AtLTI30* was widely expressed in leaves, stems, flowers, primary roots and lateral roots (**Figures [1A--E](#F1){ref-type="fig"}**). Moreover, we found that the GUS activities of *proLTI30::GUS* transgenic plants were significantly increased after dehydration stress and ABA treatments for 1, 3, and 6 h through GUS activity assay (**Figure [2A](#F2){ref-type="fig"}**). The GUS result is consistent with previous studies that ABA and dehydration induced the transcript level of *AtLTI30* using northern blot ([@B34]; [@B24]; [@B20]; [@B2]). Moreover, we also found that the transcript levels of *AtLTI30* are induced by both dehydration and ABA treatments (**Figures [2B,C](#F2){ref-type="fig"}**) using the publicly available microarray data (<http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi>) ([@B35]). These results indicate the possible link between *AtLTI30* and these stress treatments, and suggest the possible involvement of *AtLTI30* in drought stress responses in *Arabidopsis*.

![**Glucuronidase staining of *proLTI30::GUS* transgenic plants in different organs. (A)** GUS staining of 21-day-old of *proLTI30::GUS* transgenic plants. **(B--E)** GUS staining of *proLTI30::GUS* transgenic plants in leaves **(B)**, flowers **(C)**, primary root **(D)**, and lateral root **(E)**.](fpls-06-00893-g001){#F1}

![**The expression levels of *AtLTI30* in response to different stress treatments. (A)** The GUS activities of *proLTI30::GUS* transgenic plants in response to different stress treatments. For the assay, 14-day-old of *proLTI30::GUS* seedlings were subjected to dehydration (un-covering the plate in the growth chamber), or transferred to fresh MS liquid medium containing 50 μM ABA, and samples were collected at 0, 1, 3, and 6 h after treatments. The GUS activity of *proLTI30::GUS* at 0 h of treatment was normalized as 1.0. The results shown are the means ± SDs of three biological repeats, and asterisk symbols (^∗^) indicate the significant difference of *p* \< 0.05 compared to the expression at 0 h of treatment. **(B,C)** The transcript levels of *AtLTI30* in response to dehydration **(B)** and ABA **(C)** using the publicly available microarray data (<http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi>).](fpls-06-00893-g002){#F2}

Isolation of *AtLTI30* Knockout Mutants and Overexpressing Plants
-----------------------------------------------------------------

To further reveal the *in vivo* role of *AtLTI30*, we isolated the T-DNA mutants with T-DNA insertion in the extron of *AtLTI30* \[*lti30-1* (SALK_114915) and *lti30-2* (SALK_016819)\] (**Figures [3A--C](#F3){ref-type="fig"}**), and constructed *AtLTI30* overexpressing transgenic plants (**Figures [3B,C](#F3){ref-type="fig"}**). Using semi-quantitative RT-PCR analysis, the transcript of *AtLTI30* was un-detectable in *lti30-1* and *lti30-2* mutants (**Figure [3B](#F3){ref-type="fig"}**). Consistently, quantitative real-time PCR analysis also showed that the transcript level of *AtLTI30* was largely inhibited in *lti30-1* and *lti30-2* mutants, with about 30% of *AtLTI30* transcripts compared with WT plants (**Figure [3C](#F3){ref-type="fig"}**). Moreover, the *AtLTI30* overexpressing transgenic plants displayed significantly higher *AtLTI30* transcripts than WT plants, with 15--25-folds higher *AtLTI30* transcripts compared with WT, and the homozygous transgenic plants (OX-2 and OX-3) were chosen for further analysis (**Figures [3B,C](#F3){ref-type="fig"}**).

![**Characterization of *lti30* knockout mutants and *AtLTI30* overexpressing plants. (A)** Schematic diagrams of the T-DNA position of *lti30-1* and *lti30-2* mutants. **(B,C)** The transcript level of *AtLTI30* in *lti30* knockout mutants and *AtLTI30* overexpressing plants assayed by semi-quantitative RT-PCR **(B)** and quantitative real-time PCR **(C)**. The expression of *UBQ10* was used as an internal control. The results shown are the means ± SDs of three biological repeats, and asterisk symbols (^∗^) indicate the significant difference of *p* \< 0.05 in comparison to wild type (WT).](fpls-06-00893-g003){#F3}

*AtLTI30* Positively Regulates Plant Sensitivity to ABA
-------------------------------------------------------

Since the expression of *AtLTI30* was increased after ABA treatment, the responses of the WT, *lti30* knockout mutants, and *AtLTI30* overexpressing plants to ABA were further compared. Germination of *AtLTI30* overexpressing plant seeds was severely inhibited after ABA treatment, as shown with less emerged radical, less green cotyledon compared with those of WT (**Figures [4A--C](#F4){ref-type="fig"}**). On the contrary, *lti30-1* and *lti30-2* mutants showed more emerged radicals and more green cotyledons than those of WT (**Figures [4A--C](#F4){ref-type="fig"}**). These results indicate that modulation of *AtLTI30* expression positively affects ABA sensitivity in seed germination stage.

![**Modulation of *AtLTI30* expression positively affects plant sensitivity to ABA. (A)** The photograph showing 10-day-old of *Arabidopsis* plant seedlings after grown on MS plate containing different concentrations of ABA. **(B,C)** Germination rates of plant seeds on MS plate containing different concentrations of ABA that were determined as emerged radicals **(B)** and green cotyledons **(C)**. The results shown are the means ± SDs of three biological repeats, and asterisk symbols (^∗^) indicate the significant difference of *p* \< 0.05 in comparison to WT.](fpls-06-00893-g004){#F4}

*AtLTI30* Positively Regulates Drought Stress Resistance
--------------------------------------------------------

To test whether *AtLTI30* regulates drought stress resistance, 14-day-old WT, *lti30* mutants, and *AtLTI30* overexpressing plants in pots were subjected to drought stress by withholding water for 21 days and then re-watering the plants for 4 days. After the drought stress treatment, the *lti30-1* and *lti30-2* mutants displayed significantly lower survival rate, while *AtLTI30* overexpressing plants exhibited higher survival rate compared with WT (**Figures [5A,B](#F5){ref-type="fig"}**). Consistently, the *lti30-1* and *lti30-2* mutants displayed significantly higher leave water loss rate, while *AtLTI30* overexpressing plants showed significantly lower leaf water loss rate from 2 to 8 h after detachment in comparison to WT plants (**Figure [5C](#F5){ref-type="fig"}**). These results indicate that *AtLTI30* positively regulates drought stress resistance.

![***AtLTI30* positively regulates drought stress resistance in *Arabidopsis*. (A)** The photograph showing 14-day-old *Arabidopsis* plants with well-watered for 21 days, withheld water for 21 days, and re-watered for 4 days after withheld water for 21 days. **(B)** The survival rate of different genotypes after recovery for 4 days after 21 days of drought stress treatment. **(C)** Relative leaf water loss rate *in vitro* of different lines. The results shown are the means ± SDs of three biological repeats, and asterisk symbols (^∗^) indicate the significant difference of *p* \< 0.05 in comparison to WT.](fpls-06-00893-g005){#F5}

Modulation of *AtLTI30* Expression Affects H~2~O~2~ Accumulation
----------------------------------------------------------------

Oxidative burst especially H~2~O~2~ accumulation occurs following drought stress in plants. We further investigated the effects of *AtLTI30* expression on H~2~O~2~ accumulation and associated antioxidant defense enzyme activity during the drought stress treatments. During the period between 0 and 15 days of drought stress, H~2~O~2~ burst was significantly displayed in WT, the *AtLTI30* knockout mutants and overexpressing plants (**Figure [6A](#F6){ref-type="fig"}**). In comparison to WT plants, the *AtLTI30* knockout mutants showed higher levels of H~2~O~2~ at 0, 5, 10, and 15 days of drought stress, while the *AtLTI30* overexpressing plants displayed relatively lower levels of H~2~O~2~ at these periods (**Figure [6A](#F6){ref-type="fig"}**).

![**Quantifications of H~2~O~2~ content (A) and CAT activity (B) in different lines during drought stress treatments.** For the assays, 14-d-old plant leaves were subject to drought stress by withheld water for 0, 5, 10, and 15 days. The results shown are the means ± SDs of three biological repeats, and asterisk symbols (^∗^) indicate the significant difference of *p* \< 0.05 in comparison to WT.](fpls-06-00893-g006){#F6}

In accordance with the H~2~O~2~ burst, the *AtLTI30* knockout mutants exhibited lower activities of AtCATs under both control and drought stress conditions, while the *AtLTI30* overexpressing plants showed relatively higher activities of AtCATs in comparison to WT (**Figure [6B](#F6){ref-type="fig"}**). Therefore, these results indicate that the *AtLTI30* positively regulates the activities of AtCATs, and negatively regulates H~2~O~2~ accumulation during drought stress treatment.

*AtLTI30* Positively Regulates Drought Stress Resistance
--------------------------------------------------------

During the period between 0 and 15 days of drought stress, the endogenous proline content gradually increased in WT, *AtLTI30* knockout mutants and overexpressing plants (**Figure [7](#F7){ref-type="fig"}**). In comparison to WT plants, the *AtLTI30* knockout mutants exhibited lower proline contents at 0, 5, 10, and 15 days of drought stress, while the *AtLTI30* overexpressing plants displayed higher levels of proline at these periods (**Figure [7](#F7){ref-type="fig"}**).

![**Modulation of *AtLTI30* expression affects the endogenous proline level.** For the assays, 14-d-old plant leaves were subject to drought stress by withheld water for 0, 5, 10, and 15 days. The results shown are the means ± SDs of three biological repeats, and asterisk symbols (^∗^) indicate the significant difference of *p* \< 0.05 in comparison to WT.](fpls-06-00893-g007){#F7}

Discussion
==========

As a dehydrin belonging to the group II LEA family, AtLTI30/XERO2 is widely involved in plant freezing stress resistance ([@B24]; [@B20]). Not only is its transcription induced under cold stress treatment, but its overexpression confers improved freezing stress resistance in *Arabidopsis* ([@B24]; [@B20]; [@B22]). Moreover, the common expression between *CBF1/2/3* and *AtLTI30/XERO2*, and between *ABA insensitive 5* (*ABI5*) and *AtLTI30/XERO2*, indicated that *AtLTI30/XERO2* may be a direct target of both AtCBFs and AtABI5 ([@B2]). Using multiple combinations of mutations in the promoter of *AtLTI30/XERO2*, they also found that two of the ACGT and CRT/DRE elements are essential for both ABA and cold transcriptional induction of *AtLTI30/XERO2* ([@B2]). However, the possible involvement of *AtLTI30* in ABA signaling and the *in vivo* role of *AtLTI30* in plant drought stress response remains unknown.

To date, there are three effective methods to improve plant drought stress resistance. The first is to screen and identify drought tolerant varieties, and the second method is the exogenous application of multiple small molecules such as ABA, nitric oxide (NO), polyamines, hydrogen sulfide (H~2~S), and melatonin ([@B26], [@B29], [@B28]; [@B1]). The third method is the investigation of new genes that confer plant drought stress resistance and genetic breeding ([@B15]; [@B37]; [@B23]; [@B39]). In this study, the new roles of *AtLTI30* in drought stress resistance may provide a useful candidate gene for drought tolerant crop genetic breeding.

In response to drought stress, plant endogenous ABA synthesis and the following ABA-responsive genes are rapidly and largely activated. Briefly, with an increase in the endogenous ABA level, ABA receptors (Pyrabactin Resistance (PYR)/PYR1-Like (PYL)/Regulatory Components of ABA Receptor (RCAR)) disrupt the interaction between type 2C protein phosphatases (PP2Cs) and sucrose non-fermenting 1 (SNF1)-related protein kinases 2 (SnRK2s) by competitively interacting with PP2Cs. Thereafter, these interaction prevent PP2Cs-mediated dephosphorylation of SnRK2s and the activation of the SnRK2s, leading to the transcriptional activation of ABA-responsive genes ([@B8]; [@B5]; [@B11]). Moreover, ABA also induces the accumulation of H~2~O~2~, and both ABA and ABA-induced H~2~O~2~ play important roles in plant drought stress response, especially in the modulation of stomatal closure ([@B40]; [@B15]; [@B19]). In accordance with previous studies, which showed that ABA and dehydration induced the transcription level of *AtLTI30*, as evidenced by northern blot analysis ([@B34]; [@B24]; [@B20]; [@B2]), the expression of *AtLTI30* was significantly increased after ABA and drought stress treatments using *proLTI30::GUS* transgenic plants and the publicly available microarray data (<http://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi>) (**Figure [2](#F2){ref-type="fig"}**). Together with the association among ABA, H~2~O~2~ and drought stress, these results indicated the possible role of the *AtLTI30* in the process. After identifying the *AtLTI30* knockout mutants and the *AtLTI30* overexpressing plants (**Figure [3](#F3){ref-type="fig"}**), we found that the *AtLTI30* positively regulated plant sensitivity to ABA (**Figure [4](#F4){ref-type="fig"}**). In accordance with the ABA insensitive phenotype, the *lti30-1* and *lti30-2* mutants showed decreased drought stress resistance, as evidenced by a higher water loss rate and lower survival rate in comparison to WT (**Figure [5](#F5){ref-type="fig"}**). On the contrary, the *AtLTI30* overexpressing plants were more sensitive to ABA and exhibited improved drought stress resistance (**Figure [5](#F5){ref-type="fig"}**). These results suggest that *AtLTI30* may function in drought stress response in an ABA-dependent pathway. However, the stomatal response of the *AtLTI30* knockout mutants and the *AtLTI30* overexpressing plants displayed no significant difference in comparison to WT plants under mock, ABA and H~2~O~2~ conditions (Supplementary Figure [S1](#SM1){ref-type="supplementary-material"}). This result indicated that modulation of *AtLTI30* expression has no significant effect on stomatal response in *Arabidopsis*.

Reactive oxygen species (ROS) including H~2~O~2~, superoxide anions (O~2~^⋅-^), singlet oxygen (^1^O~2~) and hydroxyl radical (OH^-^) plays pivotal roles in plant drought stress responses. On one hand, H~2~O~2~ is key secondary messenger in drought stress perception and transduction ([@B40]; [@B15]; [@B19]; [@B33]). On the other hand, as toxic by-products of physiological metabolism, H~2~O~2~ accumulation is rapidly and largely increased under drought stress conditions, and overproduction of H~2~O~2~ triggers serious oxidative damage and decreased drought resistance ([@B16]; [@B17]). To cope with stress-triggered ROS overproduction and oxidative stress, plants have developed complex defense systems including both enzymatic and non-enzymatic antioxidants. Among the enzymatic enzymes, CAT catalyzes the decomposition of H~2~O~2~ into H~2~O and O~2~ and plays an essential role in controlling ROS homeostasis. In *Arabidopsis*, the *AtCATs* transcripts can be largely induced by various stress treatments including ABA, drought, salt, cold, and oxidative stresses ([@B6]; [@B14]; [@B12]). The interactions among nucleoside diphosphate kinase 2 (NDPK2), CAT2 or CAT3 and Salt Overly Sensitive 2 (SOS2) indicate the relationship between H~2~O~2~ and abiotic stress response ([@B32]). Together with previous studies showing the interaction of SOS2 and other SnRK3s with ABI1 and ABI2 ([@B10]; [@B21]) and the importance of H~2~O~2~-dependent inactivation of ABI1 and ABI2 in ABA signaling ([@B15]), CAT2 and CAT3 may occur in the same protein complex as ABI1 and ABI2, indicating the possible involvement of AtCATs in the ABA signaling pathway. In this study, the positive effect of *AtLTI30* expression on the activities of AtCATs may be directly related to drought stress-induced ROS accumulation (**Figure [6](#F6){ref-type="fig"}**), as well as *AtLTI30*-mediated drought stress resistance. Additionally, H~2~O~2~ is an important secondary messenger in ABA signal transduction ([@B15]; [@B5]). Thus, the effects of *AtLTI30* expression on ABA sensitivity, H~2~O~2~ accumulation and drought resistance further suggest the dual cross-talks among these pathways.

Based on these results, a model for *AtLTI30*-mediated drought stress response is proposed in this study (**Figure [8](#F8){ref-type="fig"}**). In response to drought stress, the endogenous ABA level is rapidly and largely induced, and thereafter induces the expression of *AtLTI30*. Firstly, overexpression of *AtLTI30* conferred ABA sensitivity, which is directly linked with ABA-mediated stress responses. Secondly, overexpression of *AtLTI30* up-regulated the activities of AtCATs, leading to less H~2~O~2~ accumulation as well as less oxidative damage under the drought stress condition. Moreover, *AtLTI30* positively regulated the endogenous level of proline, which functions as an important osmolyte in alleviating osmotic pressure under drought stress conditions ([@B27], [@B29]), thereafter resulting in less osmotic pressure in response to drought stress. Thus, the ABA sensitivity, lower H~2~O~2~ accumulation and more proline content resulted in enhanced drought stress resistance of *AtLTI30* overexpressing plants.

![**A proposed model for *AtLTI30*-mediated drought stress response in *Arabidopsis***.](fpls-06-00893-g008){#F8}

Taken together, these results indicate the possible novel role of *AtLTI30* in ABA signaling, ROS accumulation and drought stress resistance. *AtLTI30* confers enhanced drought stress resistance in *Arabidopsis*, by positively regulating ABA sensitivity, CAT activity and proline accumulation, at least partially.

Author Contributors
===================

HS conceived and directed this study, designed and performed the experiments, analyzed the data, wrote and revised the manuscript; YC and YQ performed the experiments, analyzed the data and revised the manuscript; ZC provided suggestions and revised the manuscript. All authors approved the manuscript and the version to be published, and agreed to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.

Conflict of Interest Statement
==============================

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

This research was supported by the National Natural Science Foundation of China (No.31570249) and the startup funding of Hainan University (No. kyqd1531) to HS, the Basic Research Fund of National Non-profit Research Institutions (No. CAFYBB2012043 and No. RIF2014-03) to YQ, the National Natural Science Foundation of China (No. 31370302) and "the Hundred Talents Program" (No.54Y154761O01076 and No. 29Y329631O0263) to ZC.

Supplementary Material
======================

The Supplementary Material for this article can be found online at: <http://journal.frontiersin.org/article/10.3389/fpls.2015.00893>

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Click here for additional data file.

[^1]: Edited by: *Zuhua He, Shanghai Institute for Biological Sciences, China*

[^2]: Reviewed by: *Nabil I. Elsheery, Tanta University, Egypt; Zhaoqing Chu, Shanghai Chenshan Plant Science Research Center, China*

[^3]: ^†^*These authors have contributed equally to this work.*

[^4]: This article was submitted to Plant Physiology, a section of the journal Frontiers in Plant Science
