Supachitra Chadchawan

The role of hydrogen peroxide in chitosan-induced resistance to osmotic stress in rice (Oryza sativa L.)

Chitosan is a biopolymer with multiple agricultural applications. The objective of this research was to identify the mechanism required for the chitosan response. Chitosan clearly induced resistance to osmotic stress (a surrogate for drought stress) in the ‘Leung Pratew 123’ (‘LPT123’) rice (Oryza sativa L. ‘Leung Pratew123’) by enhancing plant growth and maintenance of the photosynthetic pigments during osmotic stress, but not in the derived mutated line, LPT123-TC171. Hydrogen peroxide (H2O2) was increased after osmotic stress in both lines, but higher levels were found in the LPT123 cultivar. Chitosan application did not affect the H2O2 or glutathione content under the osmotic stress condition in the LPT123 cultivar, but decreased H2O2 accumulation in the LPT123-TC171 line. The 20-fold lower glutathione level in the LPT123 cultivar suggested a low glutathione-ascorbate cycle activity that would lead to the higher H2O2 levels. Whereas, the chitosan-mediated reduction in glutathione levels in the LPT123-TC171 line during osmotic stress suggested a higher glutathione-ascorbate cycle activity leading to low H2O2 levels. Additionally, a higher peroxidase and catalase activity following chitosan treatment of the LPT123-TC171 line supports the lower observed H2O2 level. The lipid peroxidation after osmotic stress was decreased by chitosan treatment in LPT123, but not in LPT123-TC171. The exogenous H2O2 application with chitosan treatment in LPT123-TC171 could enhance plant growth during osmotic stress. It is concluded that the limited H2O2 level, the signal molecule for chitosan responses in the LPT123-TC171 line, resulted in no beneficial effects of chitosan application for osmotic stress. Therefore, H2O2 is proposed to be one of the key components for plant growth stimulation during osmotic (drought) stress by chitosan.

The role of the OsCam1-1 salt stress sensor in ABA accumulation and salt tolerance in rice

Involvement of the salt-inducible calmodulin gene, OsCam1-1, in abscisic acid (ABA) biosynthesis during salt stress was studied in the ‘Khoa Dawk Mali 105’ (KDML105) rice cultivar (Oryza sativa L.). FL530-IL, an isogenic salt-resistant line derived from the KDML105 cultivar, accumulated a 2.9-fold higher concentration of ABA in the leaves after salt stress treatment than that for KDML105. A twenty-four and a seven- fold higher level of OsCam1-1 transcripts were detected in the leaves of the FL530-IL and KDML105 rice cultivars, respectively, after 30 min of salt stress compared to non-salt-stressed plants. Transgenic rice lines that constitutively over-express the OsCam1-1 gene were found to up-regulate ABA aldehyde oxidase and 9-cis-epoxycarotenoid dioxygenase 3, two genes involved in ABA biosynthesis, and to have a higher ABA content, when compared to the wild type and the control transgenic lines without OsCam1-1 over-expression. In addition, transgenic plants over-expressing OsCam1-1 were more tolerant to salt stress, with, for example, a better ability to maintain their shoot and root mass (as dry weight) during salt stress, than the control plants. These data indicate that OsCam1-1 signaling is likely to play an important role in ABA biosynthesis, and the level of OsCam1-1 gene expression and ABA accumulation probably contribute to salt resistance in rice.

Overexpression of a partial fragment of the salt-responsive gene OsNUC1 enhances salt adaptation in transgenic Arabidopsis thaliana and rice (Oryza sativa L.) during salt stress.

The rice (Oryza sativa L.) nucleolin gene, OsNUC1, transcripts were expressed in rice leaves, flowers, seeds and roots but differentially expressed within and between two pairs of salt-sensitive and salt-resistant rice lines when subjected to salt stress. Salt-resistant lines exhibited higher OsNUC1 transcript expression levels than salt-sensitive lines during 0.5% (w/v) NaCl salt stress for 6d. Two sizes of OsNUC1 full-length cDNA were found in the rice genome database and northern blot analysis confirmed their existence in rice tissues. The longer transcript (OsNUC1-L) putatively encodes for a protein with a serine rich N-terminal, RNA recognition motifs in the central domain and a glycine- and arginine-rich repeat in the C-terminal domain, while the shorter one (OsNUC1-S) putatively encodes for the similar protein without the N-terminus. Without salt stress, OsNUC1-L expressing Arabidopsis thaliana Atnuc1-L1 plants displayed a substantial but incomplete revertant phenotype, whereas OsNUC1-S expression only induced a weak effect. However, under 0.5% (w/v) NaCl salt stress they displayed a higher relative growth rate, longer root length and a lower H2O2 level than the wild type plants, suggesting a higher salt resistance. Moreover, they displayed elevated AtSOS1 and AtP5CS1 transcript levels. We propose that OsNUC1-S plays an important role in salt resistance during salt stress, a new role for nucleolin in plants.

Salt-responsive mechanisms in chromosome segment substitution lines of rice (Oryza sativa L. cv. KDML105)

Two chromosome segment substitution lines of Khao Dawk Mali 105 (KDML105) rice that carry quantitative trait loci for drought tolerance located on chromosome 8 (DT-QTL8) designated CSSL8-94 and CSSL8-116 were investigated for co-expression network and physiological responses to salinity compared to their parents (KDML105; drought and salt sensitive recurrent parent, and DH103; drought tolerant QTL donor). These CSSL lines show different salt-response traits under salt stress (CSSL8-94 shows higher tolerance than CSSL8-116) and possess different segments of DT-QTL8. To identify specific biological process(es) associated with salt-stress response, co-expression network analysis was constructed from each DT-QTL segment. To evaluate differential physiological mechanisms responding to salt stress, all rice lines/cultivar were grown for 21 d in soils submerged in nutrient solutions, then subjected to 150 mM NaCl for 7 d. Physiological parameters related to co-expression network analysis (photosynthetic parameters) and salt responsive parameters (Na(+)/K(+) ratio, proline content, malondialdehyde and ascorbate peroxidase activity; EC1.11.1.1) were investigated along with the expression analysis of related genes. Physiological responses under salt stress particularly photosynthesis-related parameters of CSSL8-94 were similar to DH103, whereas those of CSSL8-116 were similar to KDML105. Moreover, expression levels of photosynthesis-related genes selected from the co-expression networks (Os08g41460, Os08g44680, Os06g01850, Os03g07300 and Os02g42570) were slightly decreased or stable in CSSL8-94 and DH103 but were dramatically down-regulated in CSSL8-116 and KDML105. These differential responses may contribute to the photosynthesis systems of CSSL8-94 being less damaged under salt stress in comparison to those of CSSL8-116. It can be concluded that the presence of the specific DT-QTL8 segment in CSSL8-94 not only confers drought tolerant traits but also enhances its salt tolerant ability.

Molecular Karyotyping and Exome Analysis of Salt-Tolerant Rice Mutant from Somaclonal Variation

LPT123‐TC171 is a salt‐tolerant (ST) and drought‐tolerant (DT) rice line that was selected from somaclonal variation of the original Leuang Pratew 123 (LPT123) rice cultivar. The objective of this study was to identify the changes in the rice genome that possibly lead to ST and/or DT characteristics. The genomes of LPT123 and LPT123‐TC171 were comparatively studied at the four levels of whole chromosomes (chromosome structure including telomeres, transposable elements, and DNA sequence changes) by using next‐generation sequencing analysis. Compared with LPT123, the LPT123‐TC171 line displayed no changes in the ploidy level, but had a significant deficiency of chromosome ends (telomeres). The functional genome analysis revealed new aspects of the genome response to the in vitro cultivation condition, where exome sequencing revealed the molecular spectrum and pattern of changes in the somaclonal variant compared with the parental LPT123 cultivar. Mutation detection was performed, and the degree of mutations was evaluated to estimate the impact of mutagenesis on the protein functions. Mutations within the known genes responding to both drought and salt stress were detected in 493 positions, while mutations within the genes responding to only salt stress were found in 100 positions. The possible functions of the mutated genes contributing to salt or drought tolerance were discussed. It was concluded that the ST and DT characteristics in the somaclonal variegated line resulted from the base changes in the salt‐ and drought‐responsive genes rather than the changes in chromosome structure or the large duplication or deletion in the specific region of the genome.

Effects of chitin-rich residues on growth and postharvest quality of lettuce

Influences of the chitin-rich residues on growth and yield of Butterhead lettuce (Lactuca sativa L.) were investigated. The production and postharvest quality of lettuce were enhanced through the sole application of 5% shrimp shell powder (SSP), 20% fermented chitinous material (FCM) and 10 ml of chitinase-producing Bacillus licheniformis strain SK-1 (SK-1) or 5% SSP in combination with SK-1 and 2.5% SSP in combination with 2.5% FCM. Lettuces were cultivated during three successive crop seasons. SSP and/or FCM treatments resulted in significant increases in yield in all crop seasons. When applied in the first and third crop seasons, lettuces grown with the presence of 20% FCM showed the highest increase in yield as evaluated in terms of fresh weight, dry weight, leaf number, and leaf width and length. Weight losses after storage at 8°C and 60% relative humidity for 2 weeks were significantly reduced in all treatments with SSP or FCM. The results suggest that the application of 20% FCM is the best all-year-round supplement for Butterhead lettuce cultivation.

High Performance of Photosynthesis and Osmotic Adjustment Are Associated With Salt Tolerance Ability in Rice Carrying Drought Tolerance QTL: Physiological and Co-expression Network Analysis

Understanding specific biological processes involving in salt tolerance mechanisms is important for improving traits conferring tolerance to salinity, one of the most important abiotic stresses in plants. Under drought and salinity stresses, plants share overlapping responsive mechanisms such as physiological changes and activation of signaling molecules, which induce and transmit signals through regulator genes in a regulatory network. In this study, two near isogenic lines of rice carrying chromosome segments of drought tolerance QTL on chromosome 8 from IR68586-F2-CA-31 (DH103) in the genetic background of sensitive cultivar “Khao Dawk Mali 105; KDML105” (designated as CSSL8-94 and CSSL8-95) were used to investigate physiological responses to salt stress [namely growth, Na+/K+ ratio, water status, osmotic adjustment, photosynthetic parameters, electrolyte leakage (EL), malondialdehyde (MDA), proline and sugar accumulations], compared with the standard salt tolerant (Pokkali; PK) and their recurrent parent (KDML105) rice cultivars. Physiological examination indicated that both CSSLs showed superior salt-tolerant level to KDML105. Our results suggested that salt tolerance ability of these CSSL lines may be resulted from high performance photosynthesis, better osmotic adjustment, and less oxidative stress damage under salt conditions. Moreover, to explore new candidate genes that might take part in salt tolerance mechanisms, we performed co-expression network analysis for genes identified in the CSSL rice, and found that Os08g419090, the gene involved with tetrapyrrole and porphyrin biosynthetic process (chlorophyll biosynthetic process), Os08g43230 and Os08g43440 (encoded TraB family protein and cytochrome P450, respectively) might have unprecedented roles in salt stress tolerance.

OsNucleolin1-L Expression in Arabidopsis Enhances Photosynthesis via Transcriptome Modification under Salt Stress Conditions

OsNUC1 encodes rice nucleolin, which has been shown to be involved in salt stress responses. Expression of the full-length OsNUC1 gene in Arabidopsis resulted in hypersensitivity to ABA during germination. Transcriptome analysis of the transgenic lines, in comparison with the wild type, revealed that the RNA abundance of >1,900 genes was significantly changed under normal growth conditions, while under salt stress conditions the RNAs of 999 genes were found to be significantly regulated. Gene enrichment analysis showed that under normal conditions OsNUC1 resulted in repression of genes involved in photosynthesis, while in salt stress conditions OsNUC1 increased expression of the genes involved in the light-harvesting complex. Correspondingly, the net rate of photosynthesis of the transgenic lines was increased under salt stress. Transgenic rice lines with overexpression of the OsNUC1-L gene were generated and tested for photosynthetic performance under salt stress conditions. The transgenic rice lines treated with salt stress at the booting stage had a higher photosynthetic rate and stomatal conductance in flag leaves and second leaves than the wild type. Moreover, higher contents of Chl a and carotenoids were found in flag leaves of the transgenic rice. These results suggest a role for OsNUC1 in the modification of the transcriptome, especially the gene transcripts responsible for photosynthesis, leading to stabilization of photosynthesis under salt stress conditions.

Data in support of Photosynthetic Responses in a Chromosome Segment Substitution Line of `Khao Dawk Mali 105´ Rice at Seedling Stage

The rice chromosome segment substitution lines (CSSLs) of ‘Khao Dawk Mali 105’ (‘KDML105’) genetic background were developed by backcrossing with ‘KDML105’ rice and transferring the region from chromosome 1 of DH212 which was expected to contain the full putative salt tolerance genetic region. Line of CSSL11, CSSL12, and CSSL16 contained the full putative salt tolerance genetic region were evaluated with the parental lines, ‘KDML105’ and DH212 at seedling stage of rice. The physiological responses in rice plants were grown under normal condition and 75 mM of NaCl, and then comparative photosynthetic parameters, chlorophyll fluorescence parameters, PhiPS2, ETR, NPQ, as well as growth analysis. In this article, the data of physiological response evaluation in rice at seedling stage after salt stress treatment can be found. This can be useful as the information of the photosynthesis response to salt stress to other rice cultivars and related species.

Proteomic analysis of drought-responsive proteins in rice reveals photosynthesis-related adaptations to drought stress

Drought stress inhibits rice growth and biomass accumulation. To identify novel regulators of drought-stress responses in rice, we conducted a proteome-level study of the stress-susceptible (SS) Oryza sativa L. cv. ‘Leung Pratew 123’ and its stress-resistant (SR) somaclonal mutant line. In response to osmotic-stress treatments, 117 proteins were differentially accumulated, with 62 and 49 of these proteins detected in the SS and SR rice lines, respectively. There were six proteins that accumulated in both lines. The proteins in the SS line were mainly related to metabolic processes, whereas the proteins identified in the SR line were primarily related to retrotransposons. These observations suggest that retrotransposons may influence the epigenetic regulation of gene expression in response to osmotic stress. To identify the biological processes associated with drought tolerance in rice, we conducted a co-expression network analysis of 55 proteins that were differentially accumulated in the SR line under osmotic-stress conditions. We identified a major hub gene; LOC_Os04g38600 (encoding a glyceraldehyde-3-phosphate dehydrogenase), suggesting that photosynthetic adaptation via NADP(H) homeostasis contributes to drought tolerance in rice.