There may be some regulation controlled by a threshold value for Cd plant status, in both species. Long-term contamination with 0.1 µM would be below this threshold, thanks to the PC sequestration for example. On the other hand, long-term contamination with 10 µM would exceed this limit and result in down-regulation of the transport proteins of the HATS, possibly because of excess free Cd in the cytosol or because of some signal from the shoots. For both plants, cell wall sorption efficiency appears to be improved by the high Cd concentration in the growth solution, whereas the low concentration had generally no significant impact. The increase in the cell wall binding efficiency after high internal Cd accumulation may be related to the down-regulation of intracellular uptake. However, the reduction in symplastic influx is very low and cannot account for the increase observed at the apoplastic level. On the contrary, the up-regulation of the adsorption rate may well account for the decrease in the symplastic uptake. The apparent up-regulation of Cd binding properties may be due to modifications of the root cell-wall adsorption characteristics, particularly the root CEC. Cadmium stress is known to affect cell wall composition. First, Cd increases the proportion of acidic pectins . Secondly, the cell wall CEC may be increased through regulation of enzymes. For instance, pectinmethylesterase has been suggested to be stimulated in the outer cell wall domains of Cd-stressed plants, resulting in a strong decrease in the methylesterification of the acidic pectins. Thus, Cd strongly increases the acid pectins/esterified pectins ratio, hence the higher CEC, particularly in the middle lamellae . This low degree of esterification enhances the adsorption of all metallic trace elements , improving the plant tolerance of the metal. As dry mass did not vary with the level of contamination,40 litre pot there may be no significant difference in the proportion of young roots and then no decrease in the root CEC due to the age of roots.
Therefore, the insignificant effect of low Cd contamination on the apoplastic adsorption rate could be accounted for by the existence of some Cd-stress threshold below which there is no regulation mechanism. A long-lasting tolerance to aluminum ion is an essential phenotype for perennial plants growing on strong acid soils for longer periods. There is increasing evidence that plants with superior Al tolerances are relatively easily found in woody plants, such as tropical plantation trees Melaleuca cajuputi and Paraserianthes falcataria . Some woody plants, including tea, hydrangea, and Melastoma malabathricum, are also known as Al-accumulators that retain large amounts of Al in their above ground organs . However, the mechanisms responsible for high Al tolerance or high Al accumulation in woody plants remain to be elucidated. Our preliminary screening has successfully identified that root elongation in seedlings of Cinnamomum camphora, an evergreen tree widely distributed or planted in China and its neighboring regions, is much less inhibited even at high Al concentrations in a simple ionic solution at least for several days. Our finding is consistent with a study that reported no growth reduction in C. camphora seedlings against Al in a nutrient solution for 5 weeks . As a first step in understanding long-term Al tolerance mechanisms in seedlings of C. camphora, we employed a pulse Al exposure every two days for 60 days in measurements of root elongation and Al accumulation in each organ. To understand Al transport mechanisms in shoots, we also examined the Al accumulation patterns in branch cuttings of C. camphora. Root architecture influences nutrient and water uptake, anchorage, and mechanical support, interactions with microbes, and responses to various abiotic stress factors . Since water and mineral supply are often limited in the soil, a plant with a more extensive root system exhibits higher performance with regard to the tolerance of drought and poor nutrient conditions . Several factors, including root angle, root growth rate, and root types, influence root architecture . Root growth requires the successive formation of new cells from stem cells in the root apical meristem , and the progeny of such stem cells divide rapidly and enter the elongation/differentiation zone . To maintain root meristem activity, the rates of cell division and differentiation have to be coordinated .
Plant hormones greatly influence the balance between cell division and cell differentiation . In addition, the interaction between cytokinin and auxin determines the size of the RAM through the regulation of the genes involved in auxin signaling and/or transport to ensure an appropriate auxin gradient . The rice root system consists of one seminal root, numerous adventitious roots, and lateral roots that emerge from the other two types . Lateral roots are the major components involved in the absorption of nutrients and in interactions with the surrounding soil environment . Lateral root formation represents a complex developmental process modulated by several hormones, including auxin and ethylene . Well defined and closely coordinated cell division activities give rise to lateral root primordia . While lateral roots originate from pericycle cells adjacent to xylem poles in Arabidopsis , pericycle and endodermal cells located near phloem poles are the origins of lateral roots in rice and maize . Their development is initiated by the asymmetric division of the pericycle cells, and subsequent divisions result in the formation of dome-shaped, multilayered, lateral root primordia . After the initiation of asymmetric division, the primordia emerge, form active meristems, and break through the epidermal cells to become new lateral roots. Auxin is essential for various steps in the course of root development—from cell fate acquisition to meristem initiation, emergence, and elongation . In Arabidopsis, auxin is mainly synthesized in young apical tissues of the shoots and roots . Indole-3-acetic acid is considered the major form of auxin, with tryptophan being its precursor . Among the four pathways of IAA biosynthesis from Trp, the indole-3-pyruvic acid pathway is the major pathway in Arabidopsis . In the IPyA pathway, tryptophan aminotransferases convert Trp into IPyA, and YUCCAs synthesize IAA from IPyA, a rate-limiting step for the pathway .
In rice, FISH BONE encodes a Trp aminotransferase; loss of function results in pleiotropic abnormal phenotypes, which include small leaves with large lamina joint angles, unusual vascular development, and defects in root development, which are all consistent with a decrease in internal IAA levels . Mutations in CONSTUTIVELY WILTED1result in narrow and rolled leaves, in addition to the decreased growth of lateral and crown roots . Conversely, the over expression of OsYUC1 causes an increase in IAA accumulation, and auxin-overproducing phenotypes are observed . Such phenotypes are subject to the presence of the transcription factor WUSCHEL-RELATED HOMEOBOX 11 , a key regulator of root development . In rice, auxin induces WOX11 transcription,collection drainage which establishes the YUCCA–auxin–WOX11 module for root development . Ethylene also controls root development. Treatment with low concentrations of an ethylene precursor, 1-aminocyclopropane- 1-carboxylic acid , promotes the initiation of lateral root primordia. In contrast, exposure to higher ACC concentrations inhibits such initiation considerably, while also promoting the growth of already existing lateral root primordia . The regulation is linked tightly with auxin . For example, ethylene application results in the accumulation of auxin at the tip of Arabidopsis primary roots through the promotion of auxin synthesis mediated by WEAK ETHYLENE INSENSIVE2/ANTHRANILATE SYNTHASE α1 and WEI7/INSENSIVE2/ ANTHRANILATE SYNTHASE β1 . WEI2 and WEI7 encode the α and β subunits, respectively, of anthranilate synthase , a rate-limiting enzyme in the biosynthesis of the auxin precursor Trp . In rice, ethylene also increases endogenous IAA concentrations in the roots; however, the effect is minimized in mutants defective in YUC8/REIN7, which participates in auxin biosynthesis . The homeobox genes are critical for growth and development because they regulate cell fate and plant specificity . A family of zinc-finger homeodomain proteins has an N-terminal conserved domain containing several cysteine and histidine residues for potential zinc binding, in addition to a C-terminal domain containing a homeodomain . Most ZF-HD proteins do not have an intrinsic activation domain, which suggests that interactions with other factors are necessary for transcriptional activation . In addition, all 14 members of the ZF-HD gene family in Arabidopsis are predominantly expressed in floral tissues and play key roles in their development . One member, AtHB33, which is negatively regulated by ARF2, is required for seed germination and primary root growth . Among the 11 ZF-HD genes in rice, the over expression of OsZHD1 and OsZHD2 induces leaf curling by controlling the number and arrangement of bulliform cells . Here, we report that the over expression of OsZHD2 in rice improves root growth by enhancing meristem activity. We demonstrated that the homeobox protein elevated ethylene concentrations by increasing the transcript levels of ethylene biosynthesis genes. We further obtained ChIP assay data that revealed an interaction between OsZHD2 and the chromatin of ACS5. Analyses of transgenic rice plants carrying DR5::GUS and DR5::VENUS revealed that the expression of the DR5 reporter gene was induced following treatment with ACC, an ethylene precursor. The results suggest that OsZHD2 increases the biosynthesis of ethylene and subsequently auxin, which stimulates root growth.We isolated a rice mutant plant with an extensive root system from a population of activation tagging lines, in which the expression of a gene is enhanced by multiple copies of the 35S enhancer introduced using T-DNA .
In Line 3A-13017, the root biomass increased significantly . At 8 DAG the seminal roots were 27% longer in the activation plants than in the WT . Their lateral roots were also much longer than in the WT at a similar stage. At the upper parts of the seminal roots, the mutant lateral roots were 144% longer than those of the WT . This activation line also had more lateral roots—230 per seminal root for Line 3A-13017 versus 179 laterals per seminal root for the WT . However, the density of lateral roots did not differ significantly between the genotypes , which indicated that the increase in the number of lateral roots was largely due to the mutant plants having longer primary roots. We located T-DNA 5 kb downstream from the stop codon of OsZHD2 in the transgenic line . Its expression was significantly higher than that of the control, potentially because of the 35S enhancer elements in the T-DNA border region . We designated this activation line as OsZHD2-D.qRT-PCR analysis revealed that the expression level of OsZHD2 was significantly higher in the root tips when compared with levels in the total root . In addition, the expression level of OsZHD2 was significantly higher in the basal parts of shoots including the SAM compared with upper parts of the shoots that contain leaf blades and sheathes . RNA in situ hybridization experiments revealed that OsZHD2 transcripts were abundant in the root tip regions . Several homeobox genes have been identified as key regulators of cell proliferation and specification at the early stages of embryogenesis in plants. Among 107 homeobox genes identified in the rice genome, the expression profiles from 93 members in different tissues during various developmental stages have been analyzed . The results of the analyses revealed that OsZHD2 is highly expressed in the SAM . To evaluate whether OsZHD2 induces meristem activity, we treated seedling plants with 10 µM EdU, a thymidine analog, for 2 h to visualize the S-phase cells that actively incorporate EdU into DNA . The assay results revealed that OsZHD2-D had a higher number of S-phase cells in the RAM compared with the number of cells in the WT . The RAM region is defined based on the number of cells in a file that extend from the quiescent center to the first elongated cell . Quantifying such epidermis cells in the meristem region of lateral roots revealed that the number increased significantly in the activation line—25 versus 15 for the WT —which suggested that enhanced OsZHD2 expression led to the elongation of the RAM region.To confirm that the phenotypes observed from OsZHD2-D were due to the elevated expression levels of OsZHD2, we generated transgenic plants that expressed full-length OsZHD2 cDNA under the control of the maize Ubi promoter . From six independently transformed plants, we selected two lines, OX2 and OX4, which expressed OsZHD2 at high levels . Both had more extensive root systems compared with those of the out segregated WT . Their seminal roots and lateral roots were also significantly longer , and the plants had more lateral roots than the WT . However, the density of lateral roots did not vary among genotypes .