GhmiR858 Inhibits the Accumulation of Proanthocyanidins by Targeting GhTT2L in Cotton (Gossypium hirsutum).

Proanthocyanidins (PAs) are predominantly regulated at the transcriptional level by sophisticated regulatory networks. In cotton, the role of miRNAs as key regulatory factors at the post-transcriptional level is still unclear. Here, we demonstrated that GhmiR858 negatively regulates PA accumulation in cotton leaves and calli by targeting GhTT2L. Excessive expression of GhmiR858 restrained the expression of GhTT2L, resulting in a significant decrease in PA abundance. Conversely, a reduction in GhmiR858 activity upregulated GhTT2L, which increased PA accumulation. Additionally, GhTT2L was found to positively regulate PA accumulation in both cotton and Arabidopsis. Further analyses showed that GhTT2L interacted with transcription factor GhTTG1, which directly binds to the GhANR promoter, to facilitate its transcription. This study provides new information to guide future studies of the PA regulatory mechanisms affected by miRNAs as well as the breeding of novel varieties of colored cotton with rich PAs.

A signal R3-type, CAPRICE-like MYB transcription factor from Dendrobium nobile controls trichome and root-hair development in Arabidopsis.

The CAPRICE-like MYB transcription factors with R3 MYB motif play a central role in regulating trichome and root-hair development in plants. We identified the homologous gene of ENHANCER OF TRY AND CPC (ETC) in Arabidopsis from Dendrobium nobile Lindl with full cDNA sequence and genomic sequence (CAPRICE-LIKE MYB, DnCPL and DngCPL) respectively. Phylogenic analyses revealed a close relationship of CAPRICE-like MYB TFs between D. nobile and A. thaliana. Promoter analysis indicated that DnCPL is specifically expressed in trichome basal cells of leaf epidermis and root hairs. Overexpression of DnCPL results in the suppression of trichome formation and overproduction of root hairs. In transgenic plants overexpressing DnCPL and DngCPL, trichome formation was inhibited, moreover, no trichomes were observed in tissues of aerial parts, and root-hair differentiation was significantly enhanced by strongly repressing endogenous genes of AtCPC, AtTCL1, and AtTCL2 expression, thereby enhancing AtTRY expression. The DnCPL RNAi plants formed fewer lateral roots with a corresponding change in AtCPC, AtTCL1 and AtTCL2 expression. These results suggest that Dendrobium and Arabidopsis partially use similar transcription factors for epidermal cell differentiation and the CPC-like R3 MYB, DnCPL, may be a key common regulator of plant trichome and root-hair development. The results also provided genes and means of regulation to improve the survival ratio of artificially cultivated Dendrobium with more lateral roots.

Identification and characterization of the LDAP family revealed GhLDAP2_Dt enhances drought tolerance in cotton.

Lipid droplet-associated proteins (LDAPs) play essential roles in tissue growth and development and in drought stress responses in plants. Cotton is an important fiber and cash crop; however, the LDAP family has not been characterized in cotton. In this study, a total of 14, six, seven, and seven genes were confirmed as LDAP family members in Gossypium hirsutum, Gossypium raimondii, Gossypium arboreum, and Gossypium stocksii, respectively. Additionally, expansion in the LDAP family occurred with the formation of Gossypium, which is mirrored in the number of LDAPs found in five Malvaceae species (Gossypioides kirkii, Bombax ceiba, Durio zibethinus, Theobroma cacao, and Corchorus capsularis), Arabidopsis thaliana, and Carica papaya. The phylogenetic tree showed that the LDAP genes in cotton can be divided into three groups (I, II, and III). The analysis of gene structure and conserved domains showed that LDAPs derived from group I (LDAP1/2/3) are highly conserved during evolution, while members from groups II and III had large variations in both domains and gene structures. The gene expression pattern analysis of LDAP genes showed that they are expressed not only in the reproductive organs (ovule) but also in vegetative organs (root, stem, and leaves). The expression level of two genes in group III, GhLDAP6_At/Dt, were significantly higher in fiber development than in other tissues, indicating that it may be an important regulator of cotton fiber development. In group III, GhLDAP2_At/Dt, especially GhLDAP2_Dt was strongly induced by various abiotic stresses. Decreasing the expression of GhLDAP2_Dt in cotton via virus-induced gene silencing increased the drought sensitivity, and the over-expression of GhLDAP2_Dt led to increased tolerance to mannitol-simulated osmotic stress at the germination stage. Thus, we conclude that GhLDAP2_Dt plays a positive role in drought tolerance.

Sef1, rapid-cycling Brassica napus for large-scale functional genome research in a controlled environment.

KEY MESSAGE: We demonstrated a short-cycle B. napus line, Sef1, with a highly efficient and fast transformation system, which has great potential in large-scale functional gene analysis in a controlled environment. Rapeseed (Brassica napus L.) is an essential oil crop that accounts for a considerable share of global vegetable oil production. Nonetheless, studies on functional genes of B. napus are lagging behind due to the complicated genome and long growth cycle, this is largely due to the limited availability of gene analysis and modern genome editing-based molecular breeding. In this study, we demonstrated a short-cycle semi-winter-type Brassica napus 'Sef1' with very early-flowering and dwarf phenotype, which has great potential in large-scale indoor planting. Through the construction of an F2 population of Sef1 and Zhongshuang11, bulked segregant analysis (BSA) combined with the rape Bnapus50K SNP chip assay method was used to identify the early-flowering genes in Sef1, and a mutation in BnaFT.A02 was identified as a major locus significantly affecting the flowering time in Sef1. To further investigate the mechanism of early flowering in Sef1 and discover its potential in gene function analysis, an efficient Agrobacterium-mediated transformation system was established. The average transformation efficiency with explants of hypocotyls and cotyledons was 20.37% and 12.8%, respectively, and the entire transformation process took approximately 3 months from explant preparation to seed harvest of transformed plants. This study demonstrates the great potential of Sef1 for large-scale functional gene analysis.

Cloning and functional analysis of GhDFR1, a key gene of flavonoid synthesis pathway in naturally colored cotton.

BACKGROUND: The naturally colored brown cotton fiber is the most widely used environmentally friendly textile material, which primarily contains proanthocyanidins and their derivatives. Many structural genes in the flavonoid synthesis pathway are known to improve the genetic resources of naturally colored cotton. Among them, DFR is a crucial late enzyme to synthesis both anthocyanins and proanthocyanidins in the plant flavonoid pathway. METHODS: The protein sequences of GhDFRs were analyzed using bioinformatic tools. The expression levels of GhDFRs in various tissues and organs of upland cotton Zongxu1 (ZX1), were analyzed by quantitative real-time PCR, and the expression pattern of GhDFR1 during fiber development of white cotton and brown cotton was analyzed further. The function of GhDFR1 in NCC ZX1 was preliminarily analyzed by virus induced gene silencing (VIGS) technology. RESULTS: Bioinformatic analysis revealed that GhDFRs sequences in upland cotton genome were extremely conserved. Furthermore, evolutionary tree analysis revealed that the functions of GhDFR1 and GhDFR2, and GhDFR3 and GhDFR4, presented different and shared some similarities. Our study showed GhDFR1 and GhDFR2 were specifically expressed in fibers, while GhDFR3 and GhDFR4 were specifically expressed in petals. GhDFR1 was exclusively expressed in brown cotton fiber at various stages of development and progressively increased with the growth of fiber, but the trend of expression in white cotton was quite the opposite. We silenced GhDFR1 expression in brown cotton fiber using VIGS technology, and observed the VIGS-interference plants. After reducing the expression level of GhDFR1, the period for significant GhDFR1 expression in the developing fibers changed, reducing the content of anthocyanins, and lightening the color of mature cotton fibers. CONCLUSION: GhDFR1 was preferentially expressed in brown cotton during fiber development. The timing of GhDFR1 expression for flavonoid synthesis altered, resulting in anthocyanin contents reduced and the fiber color of the GhDFR1i lines lightened. These findings showed the role of GhDFR1 in fiber coloration of NCC and provided a new candidate for NCC genetic improvement.

The cotton pectin methyl esterase gene GhPME21 functions in microspore development and fertility in Gossypium hirsutum L.

Pectin widely exists in higher plants' cell walls and intercellular space of higher plants and plays an indispensable role in plant growth and development. We identified 55 differentially expressed genes related to pectin degradation by transcriptomic analysis in the male sterile mutant, ms1. A gene encoding pectin methylesterase (GhPME21) was found to be predominantly expressed in the developing stamens of cotton but was significantly down-regulated in ms1 stamens. The tapetal layer of GhPME21 interfered lines (GhPME21i) was significantly thickened compared to that of WT at the early stage; anther compartment morphology of GhPME21i lines was abnormal, and the microspore wall was broken at the middle stage; Alexander staining showed that the pollen grains of GhPME21i lines differed greatly in volume at the late stage. The mature pollen surfaces of GhPME21i lines were deposited with discontinuous and broken sheets and prickles viewed under SEM. Fewer pollen tubes were observed to germinate in vitro in GhPME21i lines, while tiny of those in vivo were found to elongate to the ovary. The seeds harvested from GhPME21i lines as pollination donors were dry and hollow. The changes of phenotypes in GhPME21i lines at various stages illustrated that the GhPME21 gene played a vital role in the development of cotton stamens and controlled plant fertility by affecting stamen development, pollen germination, and pollen tube elongation. The findings of this study laid the groundwork for further research into the molecular mechanisms of PMEs involved in microspore formation and the creation of cotton male sterility materials.

An R2R3 MYB gene GhMYB3 functions in drought stress by negatively regulating stomata movement and ROS accumulation.

MYB transcription factors are one of the largest TF families involved in plant growth and development as well as biotic and abiotic stresses. In this study, we report the identification and functional characterization of a stress-responsive MYB gene (GhMYB3) from drought stress related transcriptome of upland cotton. GhMYB3, belonging to the R2R3-type, has high sequence similarity with AtMYB3 and was localized in the nucleus. Silence of GhMYB3 enhanced the drought tolerance of cotton seedlings and plants, reduced the water loss rate, and enhanced stomatal closure. In addition, GhMYB3i lines exhibited less ROS accumulation, as well as higher antioxidant enzyme activity and increased content of anthocyanins and proanthocyanidins than WT plants after drought stress. The expression level of flavonoid biosynthesis- and stress-related genes were up-regulated in GhMYB3i lines under drought stress condition. These results demonstrated that GhMYB3 acted as a negative regulator in upland cotton response to drought stress by regulating stomatal closure and ROS accumulation.

Identification and Functional Analysis of the Promoter of a Leucoanthocyanidin Reductase Gene from Gossypium hirsutum.

Leucoanthocyanidin reductase (LAR) is the critical enzyme in the synthesis pathway of proanthocyanidins, which are the primary pigments in brown cotton fibers. Our previous study has revealed significant differences in the expression levels of GhLAR1 between white and brown cotton fibers at 10 DPA. In this work, the expression pattern of the GhLAR1 gene was further studied, and the promoter of GhLAR1 (1780 bp) was isolated and characterized. Bioinformatic analysis indicated that GhLAR1 promoter contained many known light response elements and several defenses related to transcriptional factor-binding boxes, which may partially explain the response of the GhLAR1 to temperature, NaCl, and PEG treatments. Furthermore, GhLAR1 was preferentially and strongly expressed in fibers and flowers of cotton, and the expression levels in all tested tissues (especially fibers) of brown cotton were significantly higher than those in white cotton. Consistent with the expression analysis, the GhLAR1 promoter mainly drove GUS expression in epidermal trichomes and floral organs.

Integrative Analysis of Expression Profiles of mRNA and MicroRNA Provides Insights of Cotton Response to Verticillium dahliae.

Cotton Verticillium wilt, caused by the notorious fungal phytopathogen Verticillium dahliae (V. dahliae), is a destructive soil-borne vascular disease and severely decreases cotton yield and quality worldwide. Transcriptional and post-transcriptional regulation of genes responsive to V. dahliae are crucial for V. dahliae tolerance in plants. However, the specific microRNAs (miRNAs) and the miRNA/target gene crosstalk involved in cotton resistance to Verticillium wilt remain largely limited. To investigate the roles of regulatory RNAs under V. dahliae induction in upland cotton, mRNA and small RNA libraries were constructed from mocked and infected roots of two upland cotton cultivars with the V. dahliae-sensitive cultivar Jimian 11 (J11) and the V. dahliae-tolerant cultivar Zhongzhimian 2 (Z2). A comparative transcriptome analysis revealed 8330 transcripts were differentially expressed under V. dahliae stress and associated with several specific biological processes. Moreover, small RNA sequencing identified a total of 383 miRNAs, including 330 unique conserved miRNAs and 53 novel miRNAs. Analysis of the regulatory network involved in the response to V. dahliae stress revealed 31 differentially expressed miRNA−mRNA pairs, and the up-regulation of GhmiR395 and down-regulation of GhmiR165 were possibly involved in the response to V. dahliae by regulating sulfur assimilation through the GhmiR395-APS1/3 module and the establishment of the vascular pattern and secondary cell wall formation through GhmiR165-REV module, respectively. The integrative analysis of mRNA and miRNA expression profiles from upland cotton lays the foundation for further investigation of regulatory mechanisms of resistance to Verticillium wilt in cotton and other crops.

Function deficiency of GhOMT1 causes anthocyanidins over-accumulation and diversifies fibre colours in cotton (Gossypium hirsutum).

Naturally coloured cotton (NCC) fibres need little or no dyeing process in textile industry to low-carbon emission and are environment-friendly. Proanthocyanidins (PAs) and their derivatives were considered as the main components causing fibre coloration and made NCCs very popular and healthy, but the monotonous fibre colours greatly limit the wide application of NCCs. Here a G. hirsutum empurpled mutant (HS2) caused by T-DNA insertion is found to enhance the anthocyanidins biosynthesis and accumulate anthocyanidins in the whole plant. HPLC and LC/MS-ESI analysis confirmed the anthocyanidins methylation and peonidin, petunidin and malvidin formation are blocked. The deficiency of GhOMT1 in HS2 was associated with the activation of the anthocyanidin biosynthesis and the altered components of anthocyanidins. The transcripts of key genes in anthocyanidin biosynthesis pathway are significantly up-regulated in HS2, while transcripts of the genes for transport and decoration were at similar levels as in WT. To investigate the potential mechanism of GhOMT1 deficiency in cotton fibre coloration, HS2 mutant was crossed with NCCs. Surprisingly, offsprings of HS2 and NCCs enhanced PAs biosynthesis and increased PAs levels in their fibres from the accumulated anthocyanidins through up-regulated GhANR and GhLAR. As expected, multiple novel lines with improved fibre colours including orange red and navy blue were produced in their generations. Based on this work, a new strategy for breeding diversified NCCs was brought out by promoting PA biosynthesis. This work will help shed light on mechanisms of PA biosynthesis and bring out potential molecular breeding strategy to increase PA levels in NCCs.

Biochemical and Expression Analyses Revealed the Involvement of Proanthocyanidins and/or Their Derivatives in Fiber Pigmentation of Gossypium stocksii.

The wild cotton species Gossypium stocksii produces a brown fiber that provides a valuable resource for the color improvement of naturally colored cotton (NCC) fiber. However, the biochemical basis and molecular mechanism of its fiber pigmentation remain unclear. Herein, we analyzed the dynamics of proanthocyanidins (PAs) accumulation in developing the fiber of G. stocksii, which suggested a similar role of PAs and/or their derivatives in the fiber coloration of G. stocksii. In addition, comparative transcriptomics analyses revealed that the PA biosynthetic genes were expressed at higher levels and for a longer period in developing fibers of G. stocksii than G. arboreum (white fiber), and the transcription factors, such as TT8, possibly played crucial regulatory roles in regulating the PA branch genes. Moreover, we found that the anthocyanidin reductase (ANR) was expressed at a higher level than the leucoanthocyanidin reductases (LARs) and significantly upregulated during fiber elongation, suggesting a major role of ANR in PA synthesis in G. stocksii fiber. In summary, this work revealed the accumulation of PAs and the expression enhancement of PA biosynthetic genes in developing fibers of G. stocksii. We believe this work will help our understanding of the molecular mechanisms of cotton fiber coloration and further promote the future breeding of novel NCCs.

Identification and functional analysis of a pollen fertility-associated gene GhGLP4 of Gossypium hirsutum L.

KEY MESSAGE: Cotton male fertility-associated gene GhGLP4, encoding a germin-like protein, is essential for anthers development by keeping ROS homeostasis through reducing H2O2 level. Utilization of heterosis is an important way to increase cotton yield and improve fiber quality in hybrid cotton development programs. Male sterility is used in the development of cotton hybrids to reduce the cost of hybrid seed production by eliminating the process of emasculation. From the transcriptome analysis of genic male sterile mutant (ms1) and its background C312 of G. hirsutum, a gene encoding germin-like protein (GhGLP4) was found significantly down-regulated in different developmental stages of ms1 anthers. To explore the gene function in cotton fertility, GhGLP4 was further studied and interfered by virus-induced gene silencing. In the GhGLP4 interfered cotton lines, the expression level of GhGLP4 was significantly decreased in the stamens, and the down-regulation of GhGLP4 resulted in pollen sac closure, stigma exertion, filament shortening, decrease in the number of anthers and complete male sterility. The expression levels of respiratory burst oxidase homologs (Rboh, NADPH oxidase) were significantly altered. Further investigation showed that the SOD activity decreased while the H2O2 content increased in the atypical stamens. These results indicated that GhGLP4 gene affected the cotton anther development through maintenance of ROS homeostasis by H2O2 reduction.

[Progress in the genetic modification of blue flowers based on anthocyanin metabolism].

As water-soluble, natural pigments, anthocyanins are responsible for the red, purple and blue colors of many flowers, which attract pollinators to spread pollen. The colors of flowers are also essential for plants to survive in the nature and become one of the most significant characteristics of ornamental plants. In the booming floriculture industry, to produce various flower colors could increase the richness of natural colors, but it is still difficult to breed flowers with coveted blue color. The diversity of flower color is mainly determined by the types and contents of anthocyanins and their derivatives. The synthesis of delphinidin pigments is the key factor for breeding blue flowers. However, there are no structural genes in many plants to biosynthesize delphinidin pigments. Blue flowers are successfully created by genetic engineering in recent years. In this paper, using common ornamental plants as examples, we review the mechanism of plant flower coloration from the aspects of the key factors affecting the synthesis of delphinidin pigment and the production strategies of blue flowers based on the regulation of anthocyanin metabolism. Different strategies of molecular breeding could provide opportunities to improve colors of other floriculture plants and to develop anthocyanin-rich economic crops, such as colored cotton with blue fibers.

Genome-wide identification of cold responsive transcription factors in Brassica napus L.

BACKGROUND: Cold stress is one of the primary environmental factors that affect plant growth and productivity, especially for crops like Brassica napus that live through cold seasons. Till recently, although a number of genes and pathways involved in B. napus cold response have been revealed by independent studies, a genome-wide identification of the key regulators and the regulatory networks is still lack. In this study, we investigated the transcriptomes of cold stressed semi-winter and winter type rapeseeds in short day condition, mainly with the purpose to systematically identify the functional conserved transcription factors (TFs) in cold response of B. napus. RESULTS: Global modulation of gene expression was observed in both the semi-winter type line (158A) and the winter type line (SGDH284) rapeseeds, in response to a seven-day chilling stress in short-day condition. Function analysis of differentially expressed genes (DEGs) revealed enhanced stresses response mechanisms and inhibited photosynthesis in both lines, as well as a more extensive inhibition of some primary biological processes in the semi-winter type line. Over 400 TFs were differentially expressed in response to cold stress, including 56 of them showed high similarity to the known cold response TFs and were consistently regulated in 158A and SGDH284, as well as 25 TFs which targets were over-represented in the total DEGs. A further investigation based on their interactions indicated the critical roles of several TFs in cold response of B. napus. CONCLUSION: In summary, our results revealed the alteration of gene expression in cold stressed semi-winter and winter ecotype B. napus lines and provided a valuable collection of candidate key regulators involved in B. napus response to cold stress, which could expand our understanding of plant stress response and benefit the future improvement of the breed of rapeseeds.

Correction to: Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

In the original publication of this article [1], the authors pointed out the Fig. 4b was same with Fig. 4c. The correct Fig. 4b should be below.

Functional analysis of GhCHS, GhANR and GhLAR in colored fiber formation of Gossypium hirsutum L.

BACKGROUND: The formation of natural colored fibers mainly results from the accumulation of different anthocyanidins and their derivatives in the fibers of Gossypium hirsutum L. Chalcone synthase (CHS) is the first committed enzyme of flavonoid biosynthesis, and anthocyanidins are transported into fiber cells after biosynthesis mainly by Anthocyanidin reductase (ANR) and Leucoanthocyanidin reductase (LAR) to present diverse colors with distinct stability. The biochemical and molecular mechanism of pigment formation in natural colored cotton fiber is not clear. RESULTS: The three key genes of GhCHS, GhANR and GhLAR were predominantly expressed in the developing fibers of colored cotton. In the GhCHSi, GhANRi and GhLARi transgenic cottons, the expression levels of GhCHS, GhANR and GhLAR significantly decreased in the developing cotton fiber, negatively correlated with the content of anthocyanidins and the color depth of cotton fiber. In colored cotton Zongxu1 (ZX1) and the GhCHSi, GhANRi and GhLARi transgenic lines of ZX1, HZ and ZH, the anthocyanidin contents of the leaves, cotton kernels, the mixture of fiber and seedcoat were all changed and positively correlated with the fiber color. CONCLUSION: The three genes of GhCHS, GhANR and GhLAR were predominantly expressed early in developing colored cotton fibers and identified to be a key genes of cotton fiber color formation. The expression levels of the three genes affected the anthocyanidin contents and fiber color depth. So the three genes played a crucial part in cotton fiber color formation and has important significant to improve natural colored cotton quality and create new colored cotton germplasm resources by genetic engineering.

Nitrate Transporter 1.1 is involved in regulating flowering time via transcriptional regulation of FLOWERING LOCUS C in Arabidopsis thaliana.

Nitrate Transporter 1.1 (NRT1.1) is a nitrate transporter and sensor that modulates plant metabolism and growth. It has previously been shown that NRT1.1 is involved in the regulation of flowering time in Arabidopsis thaliana. In this study, we mainly used genetic and molecular methods to reveal the key flowering pathway that NRT1.1 may be involved in. Mutant alleles of CO and FLC, two crucial components in the flowering pathway, were introduced into the NRT1.1 defective mutant background by crossing. When the CO mutation was introduced into chl1-5 plants, the double mutant had delayed flowering time, and the CO transcription levels did not change in the chl1-5 plants. These results indicate that the CO-dependent photoperiod may be not associated with the delayed flowering shown by chl1-5. However, FLC loss of function could rescue the late flowering phenotype of the chl1-5 mutant, and FLC expression levels significantly increased in the NRT1.1 defective mutant plants. The FT expression levels in the chl1-5flc-3 double mutant plants recovered when the FLC mutation was introduced into chl1-5 plants and the up-regulation of FLC transcripts in the chl1-5 mutant plants was not related to nitrate availability. Our findings suggest that NRT1.1 affects flowering time via interaction with the FLC-dependent flowering pathway to influence its target gene FT, and that NRT1.1 may be included in an additional signaling pathway that represses the expression of FLC in a nitrate-independent manner.

Differential transcript profiling alters regulatory gene expression during the development of Gossypium arboreum, G.stocksii and somatic hybrids.

Polyploidy or genome doubling (i.e., the presence of two or more diploid parental genome sets within an organism) are very important in higher plants. Of particular interest are the mechanisms in the new microenvironment of the common nucleus, where doubled regulatory networks interact to generate a viable genetic system capable of regulating growth, development and responses to the environment. To determine the effects of whole genome merging and doubling on the global gene expression architecture of a new polyploid, derived from protoplast fusion of the A1A1 genome of Gossypium arboreum and the E1E1 genome of Gossypium stocksii, we monitored gene expression through cDNA-AFLP in the somatic hybrids (G. arboreum + G. stocksii). The genomic expression patterns of the somatic hybrids revealed that changes in expression levels mainly involved regulatory genes (31.8% of the gene expression profiles), and the AA and EE genomes contributed equally to genome-wide expression in the newly formed AAEE genome from additivity and dominance effects. These results provide a novel perspective on polyploid gene regulation and hint at the underlying genetic basis of allopolyploid adaption in the new microenvironmental nucleus.

Biotic and Abiotic Stresses Activate Different Ca2+ Permeable Channels in Arabidopsis.

To survive, plants must respond rapidly and effectively to various stress factors, including biotic and abiotic stresses. Salinity stress triggers the increase of cytosolic free Ca2+ concentration ([Ca2+]i) via Ca2+ influx across the plasma membrane, as well as bacterial flg22 and plant endogenous peptide Pep1. However, the interaction between abiotic stress-induced [Ca2+]i increases and biotic stress-induced [Ca2+]i increases is still not clear. Employing an aequorin-based Ca2+ imaging assay, in this work, we investigated the [Ca2+]i changes in response to flg22, Pep1, and NaCl treatments in Arabidopsis thaliana. We observed an additive effect on the [Ca2+]i increase which induced by flg22, Pep1, and NaCl. Our results indicate that biotic and abiotic stresses may activate different Ca2+ permeable channels. Further, calcium signal induced by biotic and abiotic stresses was independent in terms of spatial and temporal patterning.