ABI3 plays a role in de-novo root regeneration from Arabidopsis thaliana callus cells

ABSTRACT

Developmental plasticity and the ability to regenerate organs during the life cycle are a signature feature of plant system. De novo organogenesis is a common mode of plant regeneration and may occur directly from the explant or indirectly via callus formation. It is now evident that callus formation occurs through the root development pathway. In fact, callus cells behave like a group of root primordium cells that are under the control of exogenous auxin. Presence or absence of auxin decides the subsequent fate of these cells. While in presence of external supplementation of auxin they are maintained as root primordia cells, absence of exogenous auxin induces the callus cells into patterning, differentiation and finally root emergence. Here we show that in absence of functional ABI3, a prominent member of the B3 superfamily of transcription factors, root regeneration is compromised in Arabidopsis callus cells. In culture medium free of any exogenous hormone supplementation, while adventitious root emergence and growth was prominently observed in wild type cells, no such features were observed in abi3-6 cells. Expression of auxin-responsive AUX1 and GH3 genes was significantly reduced in abi3-6 cells, indicating that auxin levels or distribution may be altered in absence of ABI3.

Introduction

Plant cells have the remarkable capability to regenerate organs and tissues throughout their life cycle. The developmental and regeneration program in plants is an outcome of interplay between different hormone signaling pathways. An overlapping web of signaling between auxin and cytokinin, for example, influences different aspects of growth and development.^1,2 Explants when cultured in medium containing auxin and cytokinin have the ability to regenerate organs like root or shoot depending on ratio of the hormones used.^3,4 During callus formation, exogenous supplementation of auxin and cytokinin leads to the characteristic continuous cell proliferation, with cells retaining their regeneration capacity irrespective of the plant organ used as explant.^5,6 Callus cells can be induced to form roots through a process called de-novo root regeneration by modulating the presence or absence of hormones in the growth medium. The regeneration process can be direct or indirect. De-novo root regeneration can be directly induced by wounding or stress treatment.^7,8 In Arabidopsis thaliana, leaf explants have been reported to form adventitious roots directly when cultured on medium without added exogenous hormones.^7,9Indirect de-novo root regeneration involves an initial step of callus formation from an explant cultured on callus induction medium (CIM) and then inducing root formation by culturing these cells in root induction medium (RIM) or in medium without any hormone supplementation.¹⁰Both direct and indirect de-novo root regeneration requires intricate molecular and cellular mechanisms involving auxin and auxin-responsive factors.^7,10-13

ABI3, a member of the B3 domain containing family of transcription factors, has long been known to be involved in several ABA-mediated developmental processes including embryogenesis.^14–16 ABI3 deletion results in highly non-dormant, under-developed seeds that are desiccation intolerant and exhibit precociously induced germination.^17–19 Earlier we have shown that ABI3 mediates dehydration stress response in Arabidopsis thaliana by regulating expression of an array of downstream genes.^20,21 Furthermore, ABI3 auto-activates its own expression during dehydration stress response primarily through the Sph/RYcis element present in its promoter region.²² In this work we show that in absence of ABI3, indirect de-novo root regeneration is compromised in Arabidopsis thaliana callus cells, indicating the role of ABI3 in root regeneration.

Results and discussion

Here we report an interesting phenomenon that was observed while working with callus generated from wild type and abi3-6 homozygous plants. abi3-6 is the most severe mutant allele of ABI3 with distinct phenotypic features.^20,23,24 As per our results, wild type and abi3-6 callus cells exhibit differential root regeneration phenotype. More precisely, indirect de-novo root regeneration observed in wild type and the mutant cells was not comparable. Callus was induced from wild type and abi3-6 leaf explants respectively, by culturing them in media containing auxin and cytokinin. Subsequently, upon transferring the callus cells to MS media without any exogenous hormone supplementation, distinct emergence of multiple adventitious roots was observed from wild type cells within 5 days of culture. Similar morphological features however were not observed in abi3-6 callus cells even after 15 days of culture (Figure 1(a)). Callus is a pluripotent mass of cells that is formed upon treating explants with high concentrations of auxin. It is now known that callus formation occurs en route of root development pathway.^6,7,25-27 The cells of callus seem to resemble the tip of a root meristem, irrespective of the explant used for callus generation. This is mainly due to expression of genes specific to root tip in planta.⁶ Previous works report the presence of Lateral Root Primordia (LRP) markers in callus cells as well.⁵ It has been observed that, mutants where initial stages of lateral root formation were affected, callus development was also impaired indicating that callus development does proceed via the root development pathway.⁶

Figure 1.

Morphological features and gene expression in wild type and abi3-6 callus cells.

Wild type and abi3-6 callus cells grown in MS medium (a) without any exogenous hormone supplementation showing presence and absence of distinct de-novo root regeneration, respectively; (b) supplemented with varying concentrations of NAA; (c) supplemented with varying concentrations of 2,4-D. (d) RT-qPCR based expression analyses of genes with RNA isolated from wild type and abi3-6 callus cells. Total RNA was isolated from callus cells using TriZol®reagent and subjected to reverse transcription PCR using RevertAid Reverse Transcriptase and oligo-dT primers. cDNA obtained was subjected to q-PCR based analysis with primers specific to AUX1, GH3.7, GH3.15, and ABI5. The data represented are a mean of three independent biological repeats with standard error bars. Statistical significance of gene expression between wild type and abi3-6was calculated using ‘ANOVA-Two factor with replication’ method of analysis and p values obtained are mentioned in the respective plots.

In Arabidopsis too, callus formation is known to occur via the rooting pathway and involves the two cell fate transition steps. Firstly, the “priming step”, where cell fate transition occurs transforming regenerating cells to founder cells and secondly, the “initiation step” where founder cells changes to callus cells. While the first phase specifically involves expression of WOX11 gene, the second phase is marked by decreased expression of WOX11 and increased expression of WOX5 and LBD16 genes.^7,12,26 In planta, the subsequent process of patterning, differentiation into root apical meristem (RAM) and further root emergence is an interplay of auxin concentrations and asymmetric distribution of auxin.^7,28-30 During callus formation, exogenous auxin from the callus inducing medium’ or CIM tends to maintain the callus cell mass in a root primordium like state. However, due to lack of uniform distribution of auxin ubiquitously in all callus cells, some cells might be induced into the patterning and differentiation phases. Subsequently, when callus cells are transferred to media without any exogenous hormones, endogenous auxin levels cause patterning of the root primordium to form RAM and finally lead to de-novo root regeneration.¹⁰

The differential de-novo root regeneration observed in wild type and abi3-6 may be attributed to differential auxin signaling in abi3-6 callus cells compared to wild type. It is possible that auxin distribution or homeostasis is affected in the mutant cells which lead to inefficient root regeneration compared to wild type. To understand whether different kinds of synthetic auxin supplementation have any effect on the observed root regeneration, wild type and abi3-6 cells were cultured in MS medium supplemented with varying concentrations of either NAA or 2,4-D, respectively. Even after 15 days of culture, supplementation of neither NAA nor 2,4-D, could induce de-novo root regeneration in either wild type or mutant cells (Figure 1(b, c)). This indicated that exogenous auxin perception was not differential in wild type or abi3-6 cells and hence was not the cause for the difference in root organogenesis observed. Moreover, this observation is in consonance with the fact that high levels of exogenous auxin tend to attribute root-primordia like features to callus cells preventing them from further patterning and subsequent differentiation.^7,12,28-30 On the contrary, endogenous auxin signaling induces patterning and differentiation of callus cells into RAM. Root regeneration thus seem to involve an intricate signaling pathway fine-tuned by tight regulation of endogenous auxin concentration in specific cells.WOX11 expression which leads to the formation of root founder cells is directly induced by auxin.⁷ A switch from WOX11 to WOX5 expression, that is essential to cause transition of the root founder cells to root primordium cells, is also mediated by high auxin levels and WOX11.¹² WOX5 expression is concomitant with the patterning of RAM and the formation of a defined stem cell niche.¹² WOX11 also regulates LBD16 expression, which gradually decreases with the progress of patterning.^10,13 This entire sequence of events in callus takes place in the absence of exogenous auxin and with regulated redistribution and homeostasis of endogenous auxin finally leading to de-novo root regeneration. To summarize, continuous presence of exogenous auxin overshadows the effect of endogenous auxin and blocks the progression of callus cells beyond root primordia stage. This is reflected in our results with callus grown in presence of NAA and 2,4 D, where root regeneration failed to occur in both wild type and mutant cells (Figure 1(b, c)). Callus cells when grown without any external auxin supplementation, reflect the effect of endogenous auxin in promoting root organogenesis and the differential morphological phenotype between wild type and mutant cells become apparent (Figure 1(a)). It can thus be concluded that endogenous auxin signaling involving any one or more parameters like auxin transport, homeostasis or biosynthesis is affected in absence of functional ABI3.

Gene expression reprogramming is integrally involved in hormone-mediated cell signaling that leads to distinct morphological manifestations. We performed a comparative expression analysis of AUX1, an auxin transporter gene along with GH3.15, and GH3.7 genes involved in auxin conjugation and maintenance of auxin homeostasis, between wild type and abi3-6 cells. Expression was also checked for ABI5, a transcription factor involved in ABA signaling pathway. AUX1 encodes for an auxin influx transporter which works to alter the levels and distribution of auxin in root. aux1 mutants show agravitropic responses and have severely reduced number of lateral root primordia.^31–33 Cytoskeletal actin rearrangement, which is an important aspect of cellular elongation during patterning, is mediated by auxin and AUX1 plays a major role in such spatial re-organization.³⁴ Our results indicate that expression of AUX1 was significantly less in abi3-6 cells compared to wild type (Figure 1(d)). Such reduced expression of AUX1 may lead to altered distribution and/or levels of auxin in abi3-6 cells compared to wild type. The GH3 family of genes that code for enzymes that catalyze the conjugation of free auxin with amino acids play a key role in maintaining auxin homeostasis in plants. The GH3 proteins modulate the pool of free auxin availability and thereby regulate downstream signaling cascade. Consequently, they affect several plant growth and developmental processes and can also contribute to stress signaling. GH3.7 is a member of the GH3 family whose expression is found to be highest in the root apex and the encoded enzyme can use chorismite as an acyl acid substrate.^35,36 AtGH3.15 codes for an acyl amido synthetase that preferentially conjugate IBA with glutamine and is highly expressed in roots and mature siliques.³⁷ In Arabidopsis, GH3.15 overexpression results in longer root growth, compared to wild type.³⁷ When tested, expression level of both the GH3 genes was found to be significantly reduced in abi3-6 cells compared to wild type (Figure 1(d)). It is thus evident that deletion of ABI3 affects expression of the above-mentionedauxin-responsive genes. This may result in altered distribution and/or levels of endogenous auxin in abi3-6 mutant cells consequently affectingde-novo root regenerationnegatively, compared to wild type. ABI5, a transcription factor containing a basic leucine zipper domain, is involved in several ABA-mediated physiological processes related to seed germination and early seedling growth. Earlier reports have shown that ABI5 works downstream of ABI3 in various signaling pathways and is involved in phytohormonal crosstalks.^38–40 In-silico analysis using PLACE⁴¹ and AGRIS^42–44 revealed that upstream promoter region of ABI5 contains AuxRE (TGTCTC) as well as ABRE (ACGTG) sequences. Comparative expression of ABI5 in wild type and abi3-6 cells revealed that ABI5 expression is severely reduced in the mutant compared to wild type (Figure 1(d)). Previous studies have indicated that ABI5 expression occurs in root tips at the seedling stage indicating its role in post-embryonic root development. ABI5 also acts as a negative regulator of lateral root development in the presence of stress.^39,45,46 Auxin and ABA are known to mediate a number of physiological responses together including lateral root development.⁴⁷ The link between auxin and ABA signaling during root development was found to be mediated by PIN1, a polar transporter of auxin and involves ABI5. Low levels of exogenous auxin upregulate PIN1 expression, which in turn regulates auxin flow into the root meristem.^48,49 Furthermore, increased expression of ABI5 lead to altered accumulation of PIN1 protein and altered number and length of root apical meristem cells.⁴⁹ Whether reduced expression of ABI5 in abi3-6 cells affects endogenous auxin distribution through altered PIN1 function or influences de-novo root regeneration through a different pathway needs further investigation.

To conclude, here we report that deletion of ABI3 affects de-novo root regeneration from callus cells when cultured under hormone-free conditions, thus indicating the role of ABI3 in root regeneration. When grown in presence of exogenous auxin supplements, no differential root organogenesis is observed between wild type and abi3-6 callus cells. The phenotypic difference observed could be attributed to altered distribution and homeostasis of endogenous auxin in absence of functional ABI3.

Methods

Wild type and abi3-6 Arabidopsis thaliana seeds were surface sterilized as per the protocol followed by Bedi et.al., 2016.²⁰ Surface sterilized seeds were allowed to germinate on MS-agar plates under sterile conditions. Post 7 days of germination, small pieces of leaves from the respective plants were excised under sterile conditions and transferred onto MS-agar plates supplemented with BAP (0.5 μg/ml), 2,4-D (1 μg/ml), NAA (1 μg/ml) and IAA (1 μg/ml). The explants were allowed to grow at 22°C in dark for around 7–10 days till conspicuous callus generation. Callus cells growing from leaf explants were routinely maintained on MS-agar plates supplemented with 2,4-D (0.5 μg/ml) and NAA (1 μg/ml), and kept at 22°C under dark conditions.

For morphological studies WT and abi3-6 callus cells were cultured on MS-agar plates without any supplemental hormones and the plates were photographed at regular intervals to check for morphological changes. Similarly, wild type and abi3-6 callus cellswere cultured on MS-agar plates containing varying concentrations of NAA (1.5, 3, 5, 10 and 20 µM) and 2,4-D (2.5, 5, 10, 20 and 30 µM) and monitored for possible morphological changes.

Total RNA was isolated from callus cells using TriZol® reagent (Invitrogen) as per the manufacturer’s protocol. The isolated total RNA was subjected to reverse transcription PCR using RevertAid Reverse Transcriptase (Thermo Scientific) and oligo-dT primers. The cDNA obtained was used for q-PCR based analysis with primers specific to AUX1, GH3.7, GH3.15, and ABI5 as per the method followed by Sengupta et.al., 2020.²¹

Funding Statement

This work was supported by the Department of Biotechnology (West Bengal), India [Grant No. 54(sanc)-BT(Estt)/RD-56/2014] to Dr. Ronita Nag Chaudhuri; Department of Science and Technology, India [DST-FIST, Grant No. SR/FST/COLLEGE-014/2010(C)] to St. Xavier’s College, Kolkata.

Authors contribution

RNC conceived and initiated the work, SSG performed the experiments, RNC and SSG wrote the manuscript.

Disclosure statement

Contents of this manuscript are solely the responsibility of the authors and do not necessarily represent the official views of the funding agency.