Root predominant overexpression of iaaM and CKX genes promotes root initiation and biomass production in citrus

Promoting initiation and biomass production of roots is significant for plant-based industries including using roots as bioreactors. Two citrus genotypes, Carrizo and US-897, were used as model plants to test the effects of root-predominantly overexpressed the iaaM (indoleacetic acid-tryptophan monooxygenase) gene and a CKX (a cytokinin oxidase/dehydrogenase) gene. The iaaM transgenic lines exhibited markedly faster root initiation, more root numbers, and higher root biomass compared to their wild-type counterparts. The transgenic iaaM + CKX plants also exhibited similar phenotypes, albeit to a lesser extent than the iaaM plants. Molecular analysis revealed an auxin-responsive CsGH3.1 gene was up-regulated in the iaaM roots and iaaM + CKX roots, and a cytokinin-responsive gene CsARR5 gene was down-regulated in the iaaM + CKX roots. Our results demonstrate that root predominant overexpression of the iaaM or both the iaaM and CKX genes drastically enhances the initiation, growth and biomass production of roots. These results provide additional support that manipulation of auxin and cytokinin levels in roots via transgenic or gene-editing technologies may benefit production of high-value secondary metabolites using roots as bioreactors and also improve rooting of recalcitrant plant species. Root-predominant overexpression of an auxin synthetic gene and a cytokinin degradation gene in citrus enhance initiation and biomass production of roots.


Introduction
Roots, 'the hidden half' of the plant, directly mediate nutrient and water uptake and thus influence overall plant growth and development (Verma et al. 2021).A robust root system provides stronger support of the plant to strong winds and floods, and enhanced tolerance to abiotic stress and biotic stress (Koevoets et al. 2016).Plant roots have also been widely used as a source of bioactive molecules that are used as pharmaceuticals, agrochemicals, flavors, fragrances, pigments, bio-pesticides, and food additives (Ardalani et al. 2021;Murthy et al. 2008).
The development of complex root architectures has been a notable outcome in the evolution and adaptation of higher plants in their surroundings, enabling their colonization of land and allowing them to meet nutrients and water requirements (Verma et al. 2021).Vegetative or clonal propagation is an important practice in horticulture and forest industries to produce large numbers of homogeneous plants efficiently and cost-effectively (Guan et al. 2015;Lakehal and Bellini 2019).Adventitious roots, which mainly arise from stems and shoots, are essential for newly propagated plants to survive and are a major component of the mature root system of many plant species.Also, adventitious root development is a crucial step in clonal multiplications, micropropagations and genetic transformation (Legué et al. 2014;Rigal et al. 2012).Often, the adventitious root initiation is a limiting factor for may plant species particularly woody species including some citrus varieties that are difficult to root (De Almeida et al. 2017;Park et al. 2017).
Current advances in plant biotechnology provide an opportunity to culture plant cells, tissues, and organs to produce many important secondary metabolites in a shorter time than whole plant cultivation in the field (Baque et al. 2012;Jeong et al. 2009a, b).Roots, particularly adventitious roots, are an excellent source of highly valuable metabolites (Hussain et al. 2022;Murthy et al. 2008).Adventitious root culture has successfully been used to scale up production of secondary metabolites for many medicinal plants, such as Panax ginseng (Jeong et al. 2006;Wang et al. 2013), Oploanax elatus (Jiang et al. 2015), Eurycoma longifolia (Fan et al. 2021), Echinacea angustifolia (Cui et al. 2013), Plantago ovata (Budzianowska et al. 2022), and Rehmannia glutinosa (Rahmat et al. 2021).Adventitious root culture has been show to produce high yield and high quality secondary metabolites (Wang et al. 2013;Wu et al. 2011).For example, when adventitious roots of Echinacea purpurea were cultured in air-lift bioreactors, both biomass and secondary compounds increased by tenfold after 4 weeks of culture (Jeong et al. 2009a, b).Adventitious roots of Astragalus membranaceus produced significantly higher total polysaccharide and total saponin content compared to three-year-old roots grown in the field (Wu et al. 2011).In addition, Wang et al. (2013) reported that the total saponin content in adventitious roots was much higher than in native ginseng cell and hairy root tissue culture.Furthermore, a cluster analysis revealed that the quality of saponins produced from adventitious roots was mostly similar to that of native ginseng, and the extracts of ginseng adventitious roots exhibited a similar cellular immunoregulatory effects on mice as those of native ginseng roots.
The development and growth of adventitious roots are complex process regulated by both environmental and endogenous factors (Agulló-Antón et al. 2014), among which the plant hormone auxin and cytokinins play a central role (Da Costa et al. 2013;Della Rovere et al. 2013;Pan et al. 2021;Pacurar et al. 2014).Auxin is an essential plant hormone involved in growth and developmental process, including but not limited to cell elongation, cell division, vascular differentiation, and root growth and development (Barbez et al. 2017;Di et al. 2021;Kasahara 2016;Li et al. 2016;Wang et al. 2008;Li and Jia 2022;Camalle et al. 2022;Niu et al. 2022).Overexpressing of YUC and TAA1 genes, which are involved in auxin biosynthesis in Arabidopsis, significantly promotes formation of adventitious and lateral roots (Mashiguchi et al. 2011).When adventitious roots are used as bioreactors, auxin has always been used to enhance initiation and biomass production of roots (Jeong et al. 2006;Wang et al. 2013;Jeong et al. 2009a, b;Cui et al. 2010).The Agrobacterium tumefaciens indoleacetic acid-tryptophan monooxygenase (iaaM) gene encodes an enzyme that catalyzes the conversion of amino acid tryptophan (Trp) into indole-3-acetamide, which is hydrolyzed to indole-3-acetic acid (IAA) in plant cells (Weijers et al. 2005;Zhai et al. 2021).Overexpression of the iaaM gene in petunia (Klee et al. 1987), tobacco cv Samsun (Romano et al. 1991), and tobacco cv Xanthi (Li et al. 2017) led to drastic increases in adventitious and lateral roots.
Cytokinins are another important regulator of adventitious root development (Agulló-Antón et al. 2014).As a negative regulator in root growth and branching, lowering cytokinin levels often lead to larger root systems (Jameson and Song 2020).With root-specific expression of a cytokinin oxidase/dehydrogenase (CKX) gene in barley, the transgenic plants developed larger root systems (Ramireddy et al. 2018).In chickpea, root-specific expression of chickpea cytokinin oxidase/dehydrogenase 6 (CaCKX6) gene enhanced root growth (Khandal et al. 2020).Root-predominant expression of AtCKX2, a cytokinin oxidase/dehydrogenase gene from Arabidopsis, in poplar enhanced the root growth of poplar trees (Li et al. 2019).
However, relatively little has been done to genetically manipulate auxin and cytokinin levels in woody plants, particularly in a root specific manner.In this study, we report production and characterization of transgenic plants that overexpress an iaaM gene and in combination with a CKX gene predominately in roots of two commercial important citrus genotypes, Carrizo and US-897.

Citrus explant preparation and Agrobacterium-mediated transformation
The Carrizo (Citrus sinensis x Poncirus trifoliata) and US-897 (C.reticulata x P. trifoliata) seeds were purchased from Lyn Citrus Seed, Inc. in Arvin, CA, USA and stored at 4 °C prior to use.Healthy seeds were selected, and their outer seed coats were carefully removed.The seeds were then sterilized by immersing them in 75% ethanol for one minute, followed by freshly prepared sodium hypochlorite solutions (1% sodium hypochlorite active gradients, pH adjusted to 7.0-7.2with 1 M HCl, and several drops of Tween-20 added before use) for 15 min and rinsing with sterilized distilled water for five to six times.
Inner seed coats were removed aseptically, and then seeds were cultured on a M519 medium (purchased on https:// phyto techl ab.com) containing 30 g/L sucrose and 7 g/L agar, with a pH 5.8 (abbreviated as MS medium later on) in a growth chamber at 28 °C in complete darkness for 3-4 weeks.Epicotyls of etiolated seedlings were cut to 1 cm in length.The EHA105 Agrobacterium harboring SbUGT ::iaaM, SbUGT ::iaaM + SbUGT ::AtCKX2, and SbUGT::GusPlus constructs were cultivated until the OD 600 around 0.6-0.7 and then resuspend in the MS liquid medium containing 50 mg/L Acetosyringone (AS).The suspended solutions were incubated at 28 °C with shaking at 200 rpm for 30 min and then used to infect the epicotyl segments for 20 min.After removal of residual liquid, the infected epicotyl segments were transferred onto the MS medium containing 50 mg/L AS.

Histochemical GUS activity assay
Sliced leaves of regenerated shoots (putative SbUGT :: iaaM or SbUGT ::iaaM + SbUGT ::AtCKX2 transgenic shoots), as well as intact rooted plants (putative SbUGT ::GusPlus transgenic plants), were histochemically stained for GUS activity by incubation in X-Gluc solutions (Zhai et al. 2021) at 37 °C overnight.After the staining, the tissues or entire plants were treated with ethanol to remove chlorophylls and other pigments, until the mesophyll tissues turned white.Visual inspection was conducted for GUS positive tissues or plants.

Characterization of root initiation and growth
Shoots approximately 1.3 cm in length from the wild-type and GUS-positive SbUGT ::iaaM and SbUGT::iaaM + SbUGT::AtCKX2 plants were cultured in MS medium supplemented with 150 mg/L timentin.After 2 months of culture, the number of roots per plant was recorded.
Based on the preliminary characterization of rooting, about 1.3 cm long shoot of wild-type, representative SbUGT::iaaM and SbUGT::iaaM + SbUGT::AtCKX2 transgenic Carrizo and US-897 lines were cultured in MS medium supplemented with 150 mg/L timentin.The number of days that the first root in each shoot could be observed were recorded, and the number of roots per plant was counted after 2 months of culture.After 6 months for Carrizo plant lines or 4 months for US-897 plant lines, dry root biomass was measured.
The Actin-qF and Actin-qR primers were used to amplify the internal reference gene ActB, a highly conserved β-actin gene in citrus, to normalize the samples.Three replicates were performed on each sample.

Activity of the SbUGT promoter in citrus and production of transgenic iaaM, iaaM + CKX Carrizo and US-897 plants
To monitor the activity of the SbUGT gene promoter, we produced transgenic Carrizo plants using the SbUGT ::GUS-Plus gene.Histochemical staining of the GUS activity in the SbUGT::GUSPlus plants revealed that the SbUGT promoter was highly active in roots (Fig. 1A).Based on the results of GUS activity staining and PCR confirmation using genomic DNA as templates (data not shown), 48 and 64 independent lines of Carrizo expressing the SbUGT ::iaaM gene and the SbUGT ::iaaM + SbUGT ::AtCKX2 genes were identified, respectively.We also produced 69 and 73 independent lines of US-897 expressing the SbUGT ::iaaM gene, the SbUGT ::iaaM + SbUGT ::AtCKX2 genes, respectively.
On average, the shoots of the Carrizo SbUGT::iaaM and the SbUGT ::iaaM + SbUGT ::AtCKX2 transgenic lines produced 3.0-and 2.3-fold more roots than these of the wild-type plants (Fig. 1C), while the US-897 iaaM and iaaM + CKX lines had 2.8-and 3.3-fold more roots than these of the US-897 wild-type stem cuttings, respectively (Fig. 1C).Furthermore, 14% and 12% of the Carrizo iaaM or iaaM + CKX overexpressing shoots had four to eight roots, whereas none of the wild-type shoots produced more than three roots per plant (Fig. 1D).For US-897, 8% and 5% of iaaM and iaaM + CKX independent transgenic lines have more than four roots, and none of the wild-type plants produced more than three roots (Fig. 1D).

Root-predominant overexpression of the iaaM or iaaM + CKX genes increases growth from root segments
Root segments of wild-type Carrizo, transgenic plants of lines SbUGT::iaaM#7, and SbUGT::iaaM + SbUGT::AtCKX2#13 were cultured in the MS medium to exam their growth rate.After an 8-week culture, 50% of the iaaM#7 and 41.7% of the iaaM + CKX#13 root segments produced new roots, while only 8.3% of the wild-type root segments developed new roots (Fig. 2A).The iaaM and iaaM + CKX root segments had 7.9-and 5.3-fold more lateral roots compared to the wild-type root segments (Data not shown), respectively.The iaaM and iaaM + CKX root segments produced nearly 18-and 24-fold more root biomass compared to the wild-type root segments (Fig. 2B).
Compared to the iaaM overexpressed root segments, the new roots produced from the iaaM + CKX root explants were 34% longer (Data not shown).Based on our observations, the root tip of new roots produced from wild-type root segments turned brown gradually and ceased growth when the root length reached to 5-6 cm after 10 weeks of culture (Data not shown).In contrast, the new root tissues produced from the iaaM#7 or iaaM + CKX#13 root segments remained healthy and continued to grow for over 6 months.These results suggest that the iaaM and iaaM + CKX root tissues possess enhanced growth rate and vigor compared to the wild-type root tissues.

Root predominant overexpression of the iaaM or iaaM + CKX genes promotes rooting from shoot explants
As shown in Fig. 3A-F and Table 1, transgenic SbUGT::iaaM #7, and SbUGT::iaaM + SbUGT::AtCKX2#13 shoots exhibited significantly faster rooting and produced more and longer roots compared to the wild-type plants.In the case of Carrizo (Fig. 3A-C), the average rooting time, the day of the first root appeared from a shoot explant, of the iaaM lines was 17 days earlier than the wild-type plants.The average rooting time of the iaaM + CKX shoots was 7 days earlier than the corresponding wild-type plants (Table 1).
After 2 months of culture, the iaaM and iaaM + CKX transgenic plants had 4.1-and 2.8-fold increases in number of roots compared to wild-type plants (Table 1).The Carrizo iaaM and iaaM + CKX transgenic lines produced roots that had 13.6-and 4.5-fold increases in total root length, respectively, than those of the wild-type plants (Data not shown).
T h e U S -8 9 7 S b U G T : : i a a M a n d SbUGT::iaaM + SbUGT::AtCKX2 shoots also exhibited enhanced rooting and root growth (Fig. 3D-F).The rooting of the US-897 iaaM and iaaM + CKX lines observed was 16 and 9 days earlier than the wild-type controls, respectively (Table 1).After 2 months of culture, the US-897 iaaM and iaaM + CKX lines produced 4.4-and 2.8-fold more roots compared to the wild-type plants, respectively (Table 1).The total root length of the US-897 iaaM were 7.1-fold longer than those of the wild-type plants, while the total root length of iaaM + CKX lines were 6.6-fold longer than that of the wild-type plants (Data not shown).For some strong expressers of the iaaM gene, we also observed that both the Carrizo and US-897 iaaM transgenic lines displayed leaf epinasty and stunted growth.However, the overexpression of the CKX along with the iaaM gene effectively neutralized the negative effects of the iaaM expression.

Root predominant overexpression of the iaaM gene and iaaM + CKX genes enhance root biomass production
The SbUGT::iaaM and SbUGT ::iaaM + SbUGT ::AtCKX2 transgenic plants exhibited drastic increase in root growth (Fig. 4).The representative iaaM transgenic lines showed 9.8-and 12.1-fold more root biomass in Carrizo and  US-897, respectively, compared to the corresponding wildtype plants (Fig. 4A, B, D, E, F, H).The negative impact of the iaaM gene overexpression on growth of aerial parts becomes more pronounced with time in culture.For some strong iaaM expressers, their aerial parts stopped to grow after 4 months, even though their roots grew vigorously and healthily (Fig. 4B, F).However, the CKX gene overexpression neutralizes the negative effects of the iaaM gene overexpression (Fig. 4G), which suggests that a combined use of both SbUGT::iaaM + SbUGT::AtCKX2 genes can enhance

Overexpression of the iaaM and CKX genes alter expression of auxin-and cytokinin-responsive genes
C a r r i z o r e p r e s e n t a t i v e S b U G T: : i a a M a n d SbUGT::iaaM + SbUGT::AtCKX2 expresser: iaaM#31, iaaM#7, iaaM + CKX#13, and iaaM + CKX#27 lines were used to determine altered expression levels of auxin-responsive gene CsGH3.1 and cytokinin-responsive CsARR5 genes.The iaaM#7 plant that showed a more pronounced root growth phenotype compared to the iaaM#31 plant, had a much higher iaaM gene expression level (Fig. 5A).Consistently, expression of the CsGH3.1 gene was up-regulated in both iaaM#7 and iaaM#31 roots, with 6.1-fold and 1.6-fold increases, respectively, suggesting an increases in endogenous auxin levels in these transgenic lines (Xiao et al. 2020).In the iaaM + CKX transgenic roots, both iaaM and CKX genes were expressed simultaneously (Fig. 5C, D).Upregulation of the CsGH3.1 gene expression was observed in both iaaM + CKX#13 and iaaM + CKX#27 transgenic roots (Fig. 5E).The CsARR5 gene, a cytokinin responsive-gene (Ye et al. 2021), was significantly down-regulated in these two transgenic lines (Fig. 5F).These results demonstrate that the overexpression of the iaaM and CKX genes leads to alterations in genes that are responsive to changes in auxin and cytokinin concentrations.

Discussion
In present study, our results showed that the root-predominant overexpression of either the iaaM gene or both the iaaM and the AtCKX2 genes under the control of the SbUGT promoter significantly enhanced initiation and biomass production of roots.The overexpression of the iaaM gene promoted root initiation as evidenced by shortened rooting time in both transgenic Carrizo and US-897 plants.Also, the root-predominant overexpression of the iaaM gene led to increases in root length and number, and therefore biomass.
We have also previously reported similar results in tobacco (Li et al. 2017).Thus, we conclude that the root-predominant overexpression of the iaaM and CKX genes should lead to enhanced initiation and more vigorous growth of roots in both herbaceous and woody species.
Roots have often been used as bioreactors for biologically active substances of high value including many pharmaceuticals (Murthy et al. 2008;Baque et al. 2012).However, efficient production of root biomass is a challenge in many plant species, particularly in some woody species (Zobayed & Saxena 2003).In addition to the increases in root biomass production from stem cuttings, under this study we have also demonstrated that isolated iaaM or iaaM + CKX overexpressing root segments can initiate new roots faster, and have drastic increases in root number and biomass.The iaaM or iaaM + CKX genes we used in this study can therefore be beneficial in enhancing initiation and biomass production of roots of plant species that are of value as bioreactors, which can be particularly beneficial for the plant species that are difficult to root and have poor root growth (Agulló-Antón et al. 2014;Cui et al. 2013).
Stem cuttings are wildly used as efficient, and cost-effective starting materials for vegetative plant propagation, especially for ornamental plants and orchard trees (Agulló-Antón  Kentelky et al. 2021;Wendling et al. 2010).Even so, it is also difficult to root some woody plants, such as Eucalyptus gunnii Hook.f. (Myrtaceae) (Di Battista et al. 2019).We have shown that root predominant overexpression of the iaaM gene can drastically enhance root initiation from stem cuttings.On the other hand, the overexpression of the iaaM gene reduced shoot growth in some of the transgenic lines with strong phenotype, likely due to high levels of auxin inhibit shoot growth.However, the inhibition of shoot growth due to overexpression of the iaaM gene can be reduced or eliminated in the transgenic plants that also expressed a CKX gene.Therefore, our results show that the CKX gene expression can neutralize the negative effects caused by the iaaM gene expression in perennial woody citrus plants, similar to the results previously reported in tobacco (Li et al. 2017).
In citrus, the most devastating disease called the greening disease (also called Huanglongbing, HLB) has caused more than 40% yield reduction in citrus planting area in Florida (Graham et al. 2020).One major symptom is that HLB reduces root growth of the infected citrus trees a couple of years before the trees are dead, suggesting weaken root system is involved in the death of infected trees (Munir et al. 2018;Wang and Trivedi 2013).Further studies have shown that more vigorous root growth can drastically delay the development of many HLB symptoms and therefore provide significant protection for the infected trees.Our results reported here strongly suggest that the iaaM + CKX transgenic citrus trees we have developed when used as rootstocks may provide an excellent component for an integrative approach for managing the HLB disease.
Due to the global warming, drought has become the major abiotic stress factor limiting plant growth and development (Ilyas et al. 2021).Significant yield losses of crop plants are caused by drought stress worldwide every year.It has also been shown that vigorous root growth can improve productivity of crop plants under drought and also non-drought conditions (Guo et al. 2023).Previously, it has been shown that overexpression of CKX gene can enhance root growth and plant's drought tolerance (Wang et al. 2020a, b).Here, our results further demonstrate that more vigorous root growth may be achieved if an iaaM gene is also overexpress in roots specifically along with overexpression of a CKX gene.
For strong iaaM expressers, we have observed reduced shoot growth and leaf epinasty similar to previously reported (Guilfoyle et al. 1992(Guilfoyle et al. , 1993;;Klee et al. 1987).Even though the SbUGT promoter is predominately active in roots, there are some activity in leaf tissues.It is possible that a more root specific expression of the iaaM gene may eliminate the effects on shoots and leaves.On the other hand, auxin could be transported upwards to shoots if elevated auxin is accumulated in roots (Zhai et al. 2021).However, the combined use of the iaaM and CKX genes as we have showed in this study can effectively reduce the negative effects observed in the transgenic plants that overexpress the iaaM gene alone.
In summary, our results have demonstrated that the rootpredominant overexpression of the iaaM gene or and CKX gene can drastically enhance root initiation and growth of citrus tree, which is the first report in woody plants.We suggest that the root-specific expression of both an iaaM and a CKX gene may be useful in genetic improvement of crop plants for various applications, such as improving rooting, vegetative propagation of reluctant woody plants, and scaling up the industrial culture of adventitious roots.

Fig. 1
Fig.1SbUGT promoter is predominantly expressed in root and rootpredominant overexpression of the iaaM or iaaM + CKX genes promotes rooting.Histochemical staining of GUS activity in a rooted SbUGT::GusPlus Carrizo plant, indicating that the SbUGT promoter is predominantly active in the root (A).In (A), bar = 1 cm.Expression of SbUGT ::iaaM or SbUGT::iaaM + SbUGT::AtCKX2 in both Carrizo and US-897 plants increased percentage of rooting (B), average root number of all transgenic lines produced (C), and percentage of all

Table 1
Rooting characteristics of transgenic Carrizo and US-897 plants (De Almeida et al. 2017;sion of the iaaM and CKX genes were compared with the corresponding citrus β-actin gene expression level in the same sample.The relative gene expression fold of the CsGH3.1 and the CsARR5 were normalized by citrus β-actin gene ActB and performed by the 2 −∆∆Ct method.N/A represents no data applicable.Bars represent standard deviation (SD).Asterisks indicate significant differences from the wild-type as determined by two-tailed Student's t test in Microsoft Excel software (*P < 0.05, **P < 0.01) et al. 2014) or for those that are difficult to propagate from seeds.The formation of adventitious roots is a critical step of vegetative propagation.For large scale vegetative propagation of plants, growth regulators and other manipulations are often used to promote root initiation(De Almeida et al. 2017; Fig. 5 Root predominant overexpression of the iaaM and iaaM + CKX genes altered expression levels of auxin-and cytokininresponsive genes.A Relative iaaM gene expression levels and B elevated expression levels of auxin-responsive CsGH3.1 gene in the 6-month-cultured iaaM Carrizo roots.Relative expression level of iaaM gene (C), CKX gene (D), auxin-responsive CsGH3.1 gene (E), and F cytokinin-responsive CsARR5 gene in 6-month-cultured Carrizo iaaM + CKX roots.The overexpression of the iaaM and CKX gene increased the expression level of auxin-responsive CsGH3.1 gene and decreased the cytokinin-responsive CsARR5 gene's expression level.