Interestingly, plants can be divided into four categories based on the quantity of B required: 1) Lactifers, contain the highest amount of B ; 2) Cole crops have the second highest B concentrations; 3) Legumes and the lily family of monocots are in the third group and 4) Graminaceous plants contain the least amount of B . When graminaceous plants flower, their B requirements increase. Now we know that, except for the lactifers, the B content of plants is closely aligned with the amount of pectin in their cell walls . Cell wall scientists have discovered that the RGII fraction of cell wall pectin contains B, and that cell wall structures in plants differ among species.Cell walls of grasses are much lower in pectin, and therefore these plants contain less B . Since each carbon fixed in photosynthesis is released to the Calvin cycle in bundle sheath cells by Mn-activated NAD-malic enzyme in this sub-type of C4 plant, perhaps the Mn requirement of these plants would be higher than that of C3 or NADP-malic enzyme plants . We tested this hypothesis by using hydroponic solutions where Mn concentrations could be carefully controlled. A survey of plant nutrient solution recipes indicated that most nutrient solutions contain around 2µM Mn. In this experiment, we compared the growth and photosynthetic rates of two NAD-malic enzyme C4 plants, Pearl millet and amaranthus, with two NADP-malic enzyme C4 plants,vertical hydroponic corn and sorghum, and two C3 plants, squash and wheat. Corn, sorghum, squash and wheat produced maximum biomass with the normal 2 µM Mn concentration in the hydroponic medium.
On the other hand, NAD-malic enzyme C4 plants, Pearl millet and amaranthus, produced maximum biomass with ~50 µM Mn in the nutrient solution . Photosynthetic rate responses of each species to nutrient solution Mn concentration were similar to their biomass responses. These results clearly show that when all of the carbon going into photosynthesis goes through a single Mn-activated enzyme, plant growth response is dependent on high levels of available Mn. Legumes are some of the highest protein crops grown, and they utilize N from the atmosphere rather that relying on N fertilizer to produce this protein. There are two major types of leguminous plants when it comes to root nodules and forms of N transported from these nodules to leaves and developing pods . There are determinate nodules, which tend to be round, with life spans of about 35 days. These are nodules formed on roots of warm season legumes and contain bacteroids that fix atmospheric N and use the fixed N to synthesize the ureide molecule, allantoate, for transport in xylem to leaves and developing pods. Allantoate contains 4N’s and 4C’s, and is a very efficient molecule for transporting N. Cool season legumes have indeterminate nodules that are elongated and often form a Y-shape. Bacteroids in these nodules fix N and, in general, synthesize the amide, asparagine, for transport in xylem to leaves and pods. Asparagine contains 2N’s and 4N’s. The fixed-N is released in leaves and developing pods of ureide-transporting legumes by an enzyme called allantoate amidohydrolase, and one interesting feature of this enzyme is that it is activated by Mn .
Therefore, according to the soil N status, a large proportion of the total N, mostly protein-N in the harvested legume, comes through this Mn-activated enzyme. As with the Mn-activated NADmalic enzyme plants, perhaps ureide-utilizing leguminous plants, like soybean, cowpea and lespedeza, will require higher Mn nutritional levels than asparagine-transporting legumes, like alfalfa and clover or all legumes grown on nitrate-N. To my knowledge, the Mn-requirements of ureide-transporting, amide-transporting and nitrate-fed leguminous plants have not be directly compared. In addition to a Mn-ureide metabolism connection, there is a Mn-bacteroid connection inside the root nodule. Bacteroids depend on their host legume for a source of energy to support the nitrogen fixation process. Although plants usually transport sucrose via the phloem from leaves to root nodules, root nodule cells metabolize the sucrose and provide bacteroids organic acids, like malate, as an energy source. Bacteroids in nodules of some species, like soybean, use the Mn-activated NAD-malic enzyme in the initial step of malate utilization . Therefore, a Mn-enzyme plays a central role in root nodule/legume N metabolism! The knowledge gained on structure and metabolism of a wide range of plant species over the past few years allows us to predict special nutrient needs. High protein plants require large quantities of K because components involved in protein synthesis must be bathed in high K concentrations in order to maintain the proper configurations. Plus, K+ is used to balance the negative charges of asparagine and glutamine in proteins produced. Plants with pectin-rich cell walls have high B contents, and thus plants with low pectin cell walls have low B contents. NAD-malic enzyme sub-type C4 plants have high Mn requirements for maximum growth and photosynthesis rates since every C fixed is released in bundle sheath cells by this Mn-activated enzyme. Based on their ureide metabolism with the Mn-activated enzyme, allantoate amidohydrolase, and with malate as the primary C source for bacteroids via NAD-malic enzyme, N-fixing soybeans may have a higher Mn requirement than nitrate-N grown plants.
In addition to these examples, there are other specific nutrient requirements that can be predicted based on our knowledge of plant structure and metabolism. Both Casparian strips and suberin lamellae, two extracellular hydrophobic barriers located in the wall of endodermal cells of the root, are thought to play important roles in restricting the free difusion of solutes and water . Casparian strips act as apoplastic barriers not only to block solutes moving into the xylem through the free space between cells, but also to prevent their back fow from the stele to the apoplast of the cortex. Suberin lamellae, due to their deposition between the endodermal plasma membrane and secondary cell wall, do not block aploplastic transport but rather limit transcellular transport of nutrients and possibly water at the endodermis. Cross talk between the Casparian strip and suberin lamellae exists, with suberin being deposited in response to disruption of Casparian strips.These extracellular barriers are therefore at a cross-road between control of mineral nutrient and water uptake. However, the mechanisms that allow plants to integrate both these barrier functions to enable the simultaneous uptake of sufcient water and mineral nutrients remain under explored. Te dirigent-like protein Enhanced Suberin1 functions in the correct formation of Casparian strips by allowing the lignin, deposited at the Casparian Strip Domain through the action of Peroxidase 64 and the Respiratory Burst Oxidase Homolog F, to form into a continuous ring. In the absence of this dirigent-like protein defective Casparian strips are formed along with enhanced and early deposition of suberin in the endodermis. A similar pattern of Casparian strip disruption and response is also observed when the Casparian Strip Domain is disrupted through the loss of Casparian Strip Domain Proteins. Tese changes lead to systematic alterations in the profle of mineral nutrients and trace elements accumulating in leaves, and this phenotype provided the frst tool for identification of genes involved in Casparian strip development. Detection of the difusible vasculature-derived peptides CASPARIAN STRIP INTEGRITY FACTORS 1 & 2 through interaction with the SCHENGEN3 receptor-like kinase is what drives this endodermal response to loss of Casparian strip integrity. Here, we report that detection of a loss of Casparian strip integrity at the root endodermis by the CIFs/SGN3 pathway leads to an integrated local and long-distance response. This response rebalances water and mineral nutrient uptake,what is vertical farming compensating for breakage of the Casparian strip apoplastic seal between the stele and the cortex. This rebalancing involves both a reduction in root hydraulic conductivity driven by deactivation of aquaporins, and limitation of ion leakage through deposition of suberin in endodermal cell walls. This local root-based response is also coupled to a reduction in water demand in the shoot driven by ABA-mediated stomatal closure.The dirigent-like protein Enhanced Suberin1 functions in the formation of Casparian strips by allowing the correct deposition of lignin at the Casparian strip domain. The enhanced deposition of suberin in the esb1-1 mutant with disrupted Casparian strips can clearly be observed using the lipophilic stain Fluorol Yellow 088 close to the root tip , and this can be quantifed by counting the number of endodermal cells afer the onset of cell expansion to the frst appearance of yellow fuorescence . This early deposition of suberin is also verifed by the clear correspondence of FY 088 staining with enhanced promoter activity of known suberin biosynthetic genes, including GPAT5 monitored through both GUS staining and GFP fuorescence , and also others through GUS staining . This is further reinforced by the enhanced expression of known suberin biosynthetic genes in esb1-1 relative to wild-type . To better understand the causal link between Casparian strip integrity and enhanced deposition of suberin, we performed a reciprocal grafing experiment that revealed that the esb1-1 mutation is only required in the root to drive enhanced deposition of suberin at the endodermis, placing the function of ESB1 and the driver for increased suberin in the same tissue . To determine the cause and effect relationship between damaged Casparian strips and enhanced suberin we carefully monitored the first appearance of both Casparian strips and enhanced suberin in esb1-1. Using lignin staining in the Casparian strip marker line pCASP1::CASP1::GFP, we are able to observe that damaged Casparian strips are visible 2.5 days after sowing .
This is at least 12hr before the first indication of enhanced suberin biosynthesis, which we monitor using promoter activity of suberin biosynthetic genes GPAT5, FAR4, FAR1 and FAR5 . This was also verified by the direct observation of suberin deposition with FY 088 . The observation that treatment with the CIF2 peptide, normally leaked from the stele through loss of Casparian strip integrity, can enhance suberin deposition in wild-type plants supports our interpretation that enhanced suberin deposition is a response to loss of integrity of the Casparian strip-based apoplastic diffusion barrier. Furthermore, loss-of-function of the receptor-like kinase SGN3, required for sensing of CIFs, blocks the enhanced deposition of suberin in esb1-1 and casp1-1casp3-1 based on a chemical analysis of suberin in esb1-1 , and also on FY 088 staining. We conclude that Casparian strip defects sensed by the CIFs/SGN3 surveillance system lead to enhanced deposition of suberin in the endodermis.Te observation that enhanced suberin is deposited as a response to loss of integrity of the endodermal-based diffusion barrier between stele and cortex, raises the question, what is the function of this increased suberin deposition? Previously, the extent of endodermal suberin has been shown to be part of the response to nutrient status. We therefore tested the selectivity to solutes σNaCl, in roots varying in the extent of suberin deposition and the functionality of Casparian strips. For this, we measured solute leakage into xylem sap of pressurized roots at increasing sodium chloride concentrations in the solution bathing the roots. Taken individually, σNaCl of roots of esb1-1, sgn3-3 and wild-type were not significantly diferent from one another , which is surprising given the disruption of the Casparian strip-based apoplastic diffusion barrier in both mutants. However, removal of suberin in esb1-1, by endodermal-specific ectopic expression of a cutinase, caused a signifcant decrease in σNaCl compared to wild-type plants , and a similar tendency when compared to esb1-1 . This supports the notion that enhanced suberin deposition at the endodermis helps prevent passive solute leakage caused by defects in the Casparian strips of the esb1-1 mutant. We also observed a significant decrease in σNaCl in the double mutant esb1-1sgn3-3 compared to both wild-type and sgn3-3 . It is known that SGN3 is required for the enhanced deposition of suberin that occurs at the endodermis in esb1-1. Our observation that removal of this enhanced suberin in esb1-1sgn3-3 decreases σNaCl further supports our conclusion that the role of this increased suberin deposition is to limit solute leakage where Casparian strip barriers are disrupted.It has also been suggested that endodermal suberin may impact water permeability, though how is still unclear. To further address the role of enhanced endodermal suberin, we investigated root hydraulic conductivity of esb1-1 and observed a significant reduction by 62% with respect to wild-type.Importantly, this difference in esb1-1 Lpr originates mainly from a reduction in an aquaporin-mediated water transport pathway.